- Email: [email protected]
Measurement of lipid turnover in response to thyrotropin-releasing hormone
100
INOSITOL PHOSPHOLIPIDS AND METABOLITES
[9]
[9] M e a s u r e m e n t o f L i p i d T u r n o v e r in R e s p o n s e to Thyrotropin-Releasing Hormone
By ATSUSHI IMAI and MARVIN C. GERSHENGORN Thyrotropin-releasing hormone (TRH), a tripeptide, binds specifically to receptors on the plasma membrane of anterior pituitary cells and rapidly stimulates the secretion of prolactin and thyrotropin (TSH). 1Because the mammalian anterior pituitary gland is composed of at least six cell types, only two of which are responsive to TRH and produce prolactin (mammotropes) or TSH (thyrotropes), the heterogeneity makes the cell population isolated from normal glands less than optimal for the study of the molecular mechanisms involved in TRH action. It is preferable to employ a homogeneous population of TRH-responsive cells so that the biochemical changes monitored upon stimulation by TRH can be presumed to occur uniformly in all cells of the type present. The most widely utilized homogeneous populations of pituitary mammotropes are GH cells, such as GH3 cells or GH4C1 cells. These are cloned cell lines derived from a rat pituitary tumor (MtT/W5) that produce prolactin and growth hormone. 2 The mechanism of TRH action in TSH-producing cells has not been as well studied as in mammotropic cells, at least partly because there is no cloned TRH-responsive, TSH-producing cell line. In many cells, including these TRH-responsive pituitary cells, the interaction of stimuli with cell surface receptors enhances the turnover of membrane phospholipids. It has been proposed that the increased metabolism of phosphoinositides constitutes a signal-transducing mechanism that is initiated by stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2], a minor but metabolically labile polyphosphoinositide, by a phospholipase C to generate two putative intracellular messengers, water-insoluble 1,2-diacylglycerol (1,2-DG), and watersoluble inositol trisphosphate (InsP3). 1,2-DG, which appears to serve as an intracellular mediator by activating protein kinase C, and InsP3, which acts by releasing Ca 2÷ from a nonmitochondrial store, may function in a coordinated or synergistic manner to activate a wide variety of cellular processes. This section describes the methodology that we have employed to study TRH stimulation ofphosphoinositide metabolism in intact GH3 cells and in membranes isolated from GH3 cells. The observations J M. C. Gershengorn, Annu. Rev. Physiol. 48, 515 (1986). 2 p. M. Hinkle and A. H. Tashjian, Jr., in "Biochemical Actions of Hormones" (G. Litwack, ed.), p. 269. Academic Press, New York, 1977.
METHODS IN ENZYMOLOGY, VOL. 141
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
[9l
LIPID TURNOVER IN RESPONSE TO T R H
101
made using a highly enriched preparation of plasma membranes appear to constitute the most convincing evidence that turnover of plasma membrane phosphoinositides is primarily stimulated during cell activation by TRH. Preparation of Radiolabeled GH3 Cells
Reagents GH3 cells (supplied by American Type Tissue Collection) Ham's F10 medium (supplied by GIBCO); supplemented with 15% horse serum and 2.5% fetal bovine serum Balanced salt solution (BSS): 135 mM NaC1, 4.5 mM KC1, 1.5 mM CaC12, 0.5 mM MgCI2, 5.6 mM glucose, and 10 mM HEPES, pH 7.4 0.02% EDTA in phosphate-buffered isotonic saline Procedure. GH3 cells are grown as monolayer cultures in 150-175 cm 2 flasks in Ham's FI0 medium supplemented with 15% horse serum and 2.5% fetal bovine serum under a humidified atmosphere of 5% CO2 and 95% air at 37°. The phospholipids can be labeled by incubating the cells in the presence of a variety of radioactive precursors including phosphate, glycerol, inositol, or various fatty acids. Cells may be labeled with any of these precursors so that the radioactivity incorporated into lipids achieves a constant specific activity or for brief periods so that the specific radioactivity in the lipids is changing. The ability to label GH3 cell lipids to constant specific activity is a great advantage of this cell system because the changes in the level of the radioactivity in a given molecular species then directly reflects changes in its mass. Prelabeling GH3 cells to a constant specific radioactivity is possible because these cells are a permanent cell line and can be grown in medium supplemented with one or more of these radiolabeled precursors for 48 hr, a time that is sufficient for the radiolabel in all major lipids to attain constant specific activity. 3,4 One microCurie of [3H]inositol, 10/zCi of [32p]phosphate, and, for example, 0.1/xCi of [3H]arachidonic acid or 0.2/xCi of [14C]stearic acid are usually added per milliliter of medium. Because [3H]inositol specifically labels the phosphoinositides, the majority of the more recent studies of the effects of TRH in GH3 cells have utilized [3H]inositol-prelabeled cells. Under these conditions, over 95% of 3H radioactivity in the lipid fraction is found in phosphatidylinositol (Ptdlns), lysophosphatidylinositol, phosphatidylinositol 4-monophosphate (PtdIns4P), and PtdIns(4,5)Pz ; there is approximately 4.5 × 103 cpm/nmol Ptdlns. After labeling, the cells are harvested 3 M. J. Rebecchi, R. N. Kolesnick, and M. C. Gershengorn, J. Biol. Chem. 258, 227 (1983). 4 M. J. Rebecchi and M. C. Gershengorn, Biochem. J. 216, 287 (1983).
102
INOSlTOL PHOSPHOLIPIDS AND METABOLITES
19]
by incubating them in 0.02% EDTA in phosphate-buffered saline for 5-10 min. The cells are then collected by centrifugation at 180 g for 5 min, resuspended in BSS (1-5 x 106 cells/0.1 ml), and allowed to incubate at 26° for 20-30 min. We study the effects of TRH on the metabolism of the phosphoinositides in GH3 cells exclusively in cells in suspension; however, other workers have utilized cells in monolayer culture and have obtained similar results. The majority of experiments with GH3 cells prelabeled under conditions such that specific radioactivity of the lipids is changing are conducted with cells labeled with [32P]phosphate: The major advantage of this experimental design is that even small changes in the metabolism of all phospholipids can be detected as these are magnified by the increasing specific radioactivity of the phosphate being incorporated. For these experiments, GH3 cells are harvested as described above and incubated in BSS (1-5 × 106 cells/0.1 ml) containing 50-100/zCi of [32P]phosphate/0.1 ml at 26 ° for 40-60 min. [32P]Phosphate in the lipid fraction is recovered mainly in the more labile phospholipids whose final synthetic step is catalyzed by a kinase, in particular, Ptdlns(4,5)P2, Ptdlns4P, and phosphatidic acid. The experimental incubations of cells prelabeled to constant specific radioactivity and of cells prelabeled briefly with [32P]phosphate are performed identically. Cells are separated from the preincubation medium by centrifugation at 180 g for 5 min, washed, and resuspended in fresh BSS without radioactive precursor (1-5 x 106 cells/0.1 ml). Portions of the cell suspension (0.09 ml) are placed in individual test tubes and 0.01 ml TRH (final concentration, 0.01 to 1000 nM) or vehicle is added. The incubation is terminated by adding 1.0 ml chloroform/methanol/concentrated HCI (I00: 100: 1, v/v/v). In some experiments with cells prelabeled with [3H]inositol, the experimental incubation is performed in BSS supplemented with 10 m M LiC1; LiCI is used to inhibit the enzyme inositol monophosphate (InsP) phosphatase that converts InsP to inositol and thereby allow for the increase in inositol phosphates caused by TRH to be more readily measured. Lipid Extraction and Analysis
Reagents Chloroform/methanol/concentrated HC1 (100: 100: 1, v/v/v) 10 m M EDTA 5 M. J. Rebecchi, M. E. Monaco, and M. C. Gershengorn, Biochem. Biophys. Res. Cornmun. 101, 124 (1981).
[9]
LIPID TURNOVER IN RESPONSE TO T R H
103
Preequilibrated upper phase: Upper phase of chloroform/methanol/ concentrated HCI/HzO/lO mM EDTA (50 : 50 : 0.5 : 10 : 2.5, v/v/v/v/v) Preequilibrated lower phase: Lower phase of above mixture Silica gel H plates Silica gel G plates Procedure. Lipids are extracted according either to the methods of Bligh and Dyer 6 or of Foich. v Acidification of the chloroform/methanol solutions and EDTA permit better recovery of polyphosphoinositides from the cells; the concentration of HC1 used does not cause hydrolysis of phospholipids. The chloroform/methanol/HC1 extract is separated into two phases by adding 0.25 ml of 10 m M EDTA. After brief centrifugation, the lower phase is removed. Two-tenths milliliter preequilibrated lower phase is used to wash the upper phase to remove residual lipids. The lower phases are combined and washed with 1 ml preequilibrated upper phase at 0-4 ° in order to remove any residual water-soluble material. The resulting chloroform phases are dried under a stream of N2 and immediately redissolved in a small volume of chloroform/methanol (1:1, v/v). Portions of the samples are applied under N2 to silica gel plates. No single thin-layer chromatographic system adequately resolves all the most important lipids. We have, therefore, used a series of different solvent systems and chromatographic plates to isolate and measure the lipids of interest. Major phospholipids are separated on silica gel H plates developed in one dimension with chloroform/methanol/acetic acid/H20 ( 5 0 : 3 0 : 8 : 4 , v/v/v/v). 8 This method is usually employed for screening changes in phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, and Ptdlns (Ptdlns is not separated from phosphatidylserine), especially in experiments in which cells are labeled with [32p]phosphate. Ptdlns can be measured using this system when the cells are labeled specifically with [3H]inositol. We use silica gel G plates developed in one dimension with chloroform/pyridine/70% formic acid (50 : 30 : 7, v/v/v) to more adequately isolate phosphatidic acid and phosphatidylethanolamine. 9 1,2-Diacylglycerol (I,2-DG), the lipid product of phosphoinositide degradation due to phospholipase C activation, is isolated from other lipid compounds either on silica gel G plates using the upper phase of ethyl acetate/2,2,4-trimethylpentane/acetic acid/H20 (90:50 : 20 : 100, v/v/v/v) ~°or on silica gel G plates impregnated with 0.4 M boric acid with a E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 7 j. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem. 226, 497 (1957). 8 V. P. Skipski, R. A. Peterson, and M. Barclay, Biochem. J. 90, 374 (1964). 9 R. V. Farese, A. M. Sabir, and R. E. Larson, J. Biol. Chem. 255, 7232 (1980). to E. G. Lapetina and P. Cuatrecasas, Biochim. Biophys. Acta 573, 394 (1979).
104
INOSITOL PHOSPHOLIPIDS AND METABOL1TES ATP
f/
\
ADP
= PtdIns.,
ATP
~ PtdIns4P 7
~
I0
InsP +
DG
C TP ~./Z'J
CDP'DG'IV
~_
~ PtdIns(4,5)P
l
9
I n s P 2 "I + DG
J PPi.
ADP
z
G
3 Inositol
[9]
1'
8
InsP3 +
DG
)
, L v PtdA
FIG. 1. Metabolic pathways ofphosphoinositides. Enzymes: (1) Ptdlns(4,5)P2 phospholipase C; (2) Ptdlns4P phospholipase C; (3) Ptdlns phospholipase C; (4) Ptdlns kinase; (5) Ptdlns4P kinase; (6) Ptdlns(4,5)P2 phosphomonoesterase; (7) Ptdlns4P phosphomonoesterase; (8) InsP3 phosphomonoesterase; (9) InsP2 phosphomonoesterase; (10) InsP phosphatase; (11) 1,2-DG kinase; (12) PA:CTP cytidyltransferase; (13) CDP-DG (CMP-PA):inositol 3-phosphatidyl transferase. Reproduced with permission from Rebecchi and Gershengorn.13~
solvent system consisting of chloroform/acetone (96:4, v/v). 11 Both of these systems yield adequate separation also of triacylglycerol, 1,3-diacylglycerol, 1-monoacylglycerol, 2-monoacylglycerol, and free fatty acids, such as arachidonic acid from phospholipids. Two-dimensional separations are necessary to resolve the majority of the phospholipids including the lysophospholipids, the products of the hydrolysis by a phospholipase A2 enzyme. There are several useful two-dimensional chromatographic systems. For example, the thin-layer plates can be either 2.5% magnesium acetate-impregnated HPTLC or silica gel 60 that are developed in the first dimension with chloroform/methanol/13.5 N NH4OH (65 : 35 : 5.5, v/v/v) and with chloroform/methanol/acetone/acetic acid/H20 (3 : 1 : 4 : 1 : 0.5, v/v/v/v/v) in the second dimension) z,13 The phospholipid pathway primarily affected by T R H in GH3 cells is the polyphosphoinositide cycle (see Fig. 1). 13a The effects of TRH on these lipids and inositol sugars are best studied in cells labeled to constant H y. Kameyama, S. Yoshioka, and Y. Nozawa, Biochirn. Biophys. Acta 665, 195 (1981). 12M. J. Broekman, J. W. Ward, and A. J. Marcus, J. Clin. lnvest, 66, 275 (1980). 13A. Imai, Y. Ishizuka, S. Nakashima, and Y. Nozawa, Arch. Biochem. Biophys. 232, 259 (1984). ~3aM. J. Rebecchi and M. C. Gershengorn, in "The Receptors" (P. M. Conn, ed.), Vol. 3, p. 173. Academic Press, New York, 1985.
[9]
LIPID TURNOVER IN RESPONSE TO T R H
1 TRH
I
I
I
~
105
I
100
~
_
,r.
8O
-o 2 E o o 60
:
40
DPI I
2O I
I
0
1
I
I
2 3 4 Time (minutes)
I
I
5
FIG. 2. Time course of the changes in the levels of the phosphoinositides induced by TRH in GH3 cells prelabeled with [3H]inositol to constant specific radioactivity. TRH (1 /~M) stimulates rapid loss by 15 sec of Ptdlns(4,5)P2 and Ptdlns4P. Initial levels of Ptdlns(4,5)P~, Ptdlns4P, and Ptdlns was 100, 150, and 5000 cpm/106 ceils, respectively. Reproduced with permission from Rebecchi and Gershengorn. 4
specific radioactivity with [3H]inositol as described above. The phosphoinositides are resolved well by sequential ascending chromatography on silica gel H plates in chloroform/methanol/4 N NH4OH (45 : 35 : 10, v/v/v) containing 2 mM cyclohexylenedinitrilotetraacetic acid (CDTA) followed by chloroform/methanol/acetic acid/H20 (50 : 30 : 8 : 4, v/v/v/v): Development with both solvent systems in the same dimension and the calcium chelator CDTA permit the highly charged polyphosphoinositides, PtdIns4P and PtdIns(4,5)P2, to migrate from the origin. The Rr values for PtdIns(4,5)P2, PtdIns4P, lysoPtdIns, and Ptdlns are 0.11, 0.29, 0.68, and 0.92, respectively. Figure 2 illustrates the results of experiments in which the effects of TRH on the phosphoinositides were measured in GH3 cells prelabeled to constant specific radioactivity with [3H]inositol.4 The rapid effects of TRH to decrease the levels of PtdIns(4,5)P2 and PtdIns4P, and more slowly of PtdIns, are apparent. Alternatively, the compounds are separated on high-performance thin-layer chromatography (HPTLC) plates impregnated with 1% potassium oxalate developed with chloroform/acetone/methanol/acetic acid/H20 (40:15 : 13 : 12 : 8, v/v/v/v/v). 14 When the fatty acid residues of the phosphoinositides are not of interest, another method is to deacylate the phosphoinositides by incubating the 14 j. Jolles, H. Zwiers, A. Dekker, K. W. A. Wirtz, and W. H. Grispen, Biochem. J. 194, 283 (1981).
106
INOSITOL PHOSPHOLIPIDS AND METABOLITES
[9]
lipid with methanolic NaOH (0.2 N) for 15 min ~5,~6and separate the resulting water-soluble glycerophosphoinositol, glycerophosphoinositol 4monophosphate, and glycerophosphoinositol 4,5-bisphosphate on small anion-exchange columns (see the section on inositol phosphate analysis). Major phospholipids and neutral lipids can be visualized by staining with iodine vapor. In GH3 cells, as in most cells, the content of polyphosphoinositides is insufficient for detection in this way unless carrier standards are added during chromatography. Another method to detect phospholipids is by autoradiography when the cells are labeled with [32p]phosphate (or by adding 32p-labeled standards). We expose the thin-layer plates to Kodak X-Omat films in their covers for 16-48 hr. This is an especially useful method to detect the endogenous polyphosphoinositides from cells that are labeled briefly with [32p]phosphate because these highly labile phospholipids incorporate a major fraction of the radiolabel in lipids under these conditions. Spots are scraped into vials and radioactivity is determined. The phospholipid phosphate is determined by measuring the phosphorus content of acid hydrolysates of chromatogram scrapings using, for example, the method of Chen et al. J7 As little as 2 nmol phosphorus can be detected by this procedure. Analysis of Inositol Phosphates Reagents
0.1 M formic acid 0.2, 0.4, and 0.7 M ammonium formate in 0.1 M formic acid AG l-X2 resin (acetate form, 200-400 mesh): Supplied by Bio-Rad 5 m M myo-inositol in 0.1 M formic acid The inositol phosphates--inositol monophosphate (InsP), inositol bisphosphate (InsP2), and inositol trisphosphate (InsP3)--are formed either directly by the phospholipase C-mediated hydrolysis of the phosphoinositides and/or by sequential dephosphorylation from InsP3 ; InsP3 is the only unique reaction product as it can be formed only from the hydrolysis of Ptdlns(4,5)P2 by a phospholipase C. The inositol phosphates and inositol can be isolated by anion exchange chromatography, electrophoresis, paper chromatography, or high-pressure liquid chromatography (HPLC). The most widely used, simple technique is chromatography, using small anion-exchange resin columns. Because anion exchange chromatography 15 R. B. Ellis, T. Galliard, and J. N. Hawthorne, Biochem. J. 88, 125 (1963). 16 R. M. C. Dawson, N. Hemington, and J. B. Davenport, Biochem. J. 84, 497 (1962). ~7p. S. Chen, Jr., T. Y. Toribora, and H, Warner, Anal. Chem. 28, 1756 (1956).
[9]
LIPID TURNOVER IN RESPONSE TO T R H
I TRH 500i
i
i
i
107
i
• [3H]l~0sit0t 1,4,5-ltiph0sphate 1
Z~ [3H]Inositol 1,4-diphosoh6te
I I
I • [3H]InOSifOJ 1" rnonophosphote/
-~ 4oo o Q) o
300 2O0 100 1"
t
~ _
J_
t
/
2 5 4 5 Time (minutes) FIG. 3. Time course of the changes in the levels of the inositol phosphates induced by TRH in GH3 cells prelabeled with [3H]inosiIol to constant specific radioactivity. TRH (1 /~M) stimulates rapid and transient increase in InsP3 and InsP2 and a slower increase in InsP. Initial levels of InsP3, InsP2, InsP, and inositol were 150,500, 2000, and 10,000 cpm/5 x 106 cells, respectively. Reproduced with permission from Rebecchi and Gershengorn. 4 0
~
does not isolate the inositol phosphates from all other small, water-soluble molecules, this analysis is primarily applied to measurements of the inositol sugars from cells prelabeled with [3H]inositol. Procedure. The water-methanol upper phase of the cell extract contains the water-soluble inositol phosphates and inositol. After removal of the interface and lower phase, the upper phase is dried under a stream of N2 and the residue is redissolved in 0.1-0.2 ml of 0.1 M formic acid. The samples are then loaded onto freshly prepared 1-ml columns of resin (AG I-X2), which is, prior to use, swollen in 0.1 M formic acid containing 5 m M myo-inositol. After the sample is applied, the column is washed with 10 to 20 ml of 0.1 M formic acid to remove inositol, which is not retained by the resin and thus appears in the column flow-through, before eluting the inositol phosphates. This wash step is important because there is a much greater amount of 3H radioactivity in inositol in these cells than in the inositol phosphates. The inositol phosphates are eluted sequentially by using 0.2 M ammonium formate/0.1 M formic acid (for InsP), 0.4 M ammonium formate/0.1 M formic acid (for InsP2), and 0.7 M ammonium formate/0.1 M formic acid (for InsP3). TMIn routine assays, 10 fractions (1 ml each) are collected for each eluent. The 3H radioactivity is measured in each fraction and the content of each inositol phosphate is calculated from the counts in each peak. Figure 3 illustrates the results of experiments, 18 C. P. Downes and R. H. Michell, Biochem. J. 198, 133 (1981).
108--
INOSITOL PHOSPHOLIPIDS AND METABOLITES
[9]
from which the lipid analyses were presented in Fig. 2, of the effects of TRH on the levels of the inositol phosphates and inositol in GH3 cells prelabeled to constant specific radioactivity with [3H]inositol. 4 TRH caused a rapid increase in the levels of InsP3 and InsP2 that was followed by a slower rise in the level of InsP. It is important to note again that this system does not isolate the inositol phosphates from other small, watersoluble molecules, such as nucleotides and free phosphate, and, therefore, improved techniques are needed. New methods would be especially useful for analysis of cells that are labeled with [32P]phosphate that would permit more detailed kinetic studies of the interconversions of the inositol phosphates.
Measurement of Phosphoinositide Phospholipases C and Kinases Associated with the Plasma Membrane
In GH3 cells, TRH causes a rapid decrease in the level of Ptdlns(4,5)P2 and Ptdlns4P (Fig. 2) and stimulates a marked, transient increase in InsP3, the unique product of phospholipase C-mediated hydrolysis of Ptdlns(4,5)P2, and in InsP2 (Fig. 3). These findings prove that TRH stimulates the phospholipase C-mediated hydrolysis of Ptdlns(4,5)P2. It is unclear whether there is direct hydrolysis of Ptdlns4P that m a y also lead to the formation of InsP2. These phenomena seem to occur in the plasma membrane. Thus, we have studied the enzyme activities catalyzing these events in preparations of membranes isolated from GH3 cells.
Preparations of Membrane Fractions from GHs Cells Reagents Lysis buffers: (A) 2 m M MgC12, 25 m M Tris-HC1, pH 6.9 (B) 0.5 m M dithiothreitol (DTT), 1 mM EGTA, 1 mM sodium bicarbonate, 10 m M HEPES, pH 7.9 30, 36, 41, 45, and 50% (w/v) sucrose in lysis buffer B Procedure. The method to prepare a crude membrane fraction from GH3 cells is essentially that described by Hinkle and Phillips.19 Cells are suspended in a volume of lysis buffer A equal to four to five times the volume of the cell pellet at 0°. After 10 min, the suspension is homogenized with a Teflon-glass homogenizer and centrifuged at 400 g for 5 min. 19p. M. Hinkle and W. J. Phillips, Proc. Natl. Acad. Sci. U.S.A. 81, 6183 (1984).
[9]
LIPID TURNOVER IN RESPONSE TO T R H
109
The supernatant is centrifuged at 7500 g for 10 min and the pellet is resuspended in assay buffer at a protein concentration of 2-3 mg/ml. 2° To prepare a highly enriched fraction of plasma membranes, the cells are suspended in a volume of lysis buffer B equal to four to five times the volume of the cell pellet. The cells are lysed by incubating them at 37° for 4.5 rain, followed by addition of 15 vol of ice-cold lysis buffer and rapid mixing on a vortex mixer for 0.5 rain at 0-4 °. The homogenate is centrifuged at 800 g for l0 rain to remove nuclei and cell debris. The supernatant is centrifuged again at 100,000 g for l hr. The resulting pellet is resuspended in 4 ml of lysis buffer B that is then layered on top of a discontinuous sucrose density gradient consisting of 30, 36, 41, 45, and 50% sucrose in lysis buffer B and centrifuged at 100,000 g for 1 hr. Five narrow bands are obtained at the interfaces. The upper two bands are collected as the plasma membrane fraction, diluted with lysis buffer, and centrifuged at 100,000 g for 1 hr. (The lower two bands are enriched in endoplasmic reticulum.) The final pellet is resuspended in lysis buffer B and used immediately or stored at - 7 0 °. The purity of the preparations can be assessed by the relative recoveries of marker enzymes. We commonly use Na +,K+-ATPase as a marker for plasma membranes, succinate dehydrogenase for mitochondria, and NADH dehydrogenase for endoplasmic reticulum. The plasma membrane fraction is enriched by approximately 20-fold in Na+,K+-ATPase activity.
Assays of Membrane-Associated Enzymes Polyphosphoinositide-Specific Phospholipase C (Steps I and 2 in Fig. 1) Final assay conditions 25 mM Tris-HC1, pH 6.9 1 mM Na2EDTA 2 mM ATP 6 mM MgCI: 2 mM dithiothreitol 1 mM ouabain 10 mM LiCI (to inhibit the conversion of InsP to inositol) The reaction is initiated by the addition of 40-60/zg membrane protein isolated from GHa cells that have been prelabeled with pH]inositol to constant specific radioactivity. Factors to be tested, such as TRH and z0 R. E. Straub and M. C. Gershengorn, J. Biol. Chem. 261, 2712 (1986).
1I0
[9]
1 N O S I T O L PHOSPHOLIPIDS A N D M E T A B O L I T E S
guanine nucleotides, are added to the buffer and the incubation is performed at 37°. The reaction is terminated by addition of chloroform/methanol/HCl, and phospholipid and inositol phosphate extractions and measurements are performed as outlined above. InsP3, InsP:, and InsP are produced. As in studies with intact cells, the observations made with this in vitro experimental design do not allow for a distinction as to whether InsP2 and InsP are formed from direct phospholipase C-mediated hydrolysis of Ptdlns4P and Ptdlns, respectively, or from dephosphorylation from InsP3. Figure 4 illustrates the results of an experiment that measured the effects of TRH and GTP on the formation of InsP3 and lnsP2.20 TRH and GTP caused rapid increases in the content of both inositol polyphosphates. The level of InsP3 decreased with time, suggesting the presence of an InsPs degrading enzyme(s). The effects of TRH and GTP when added simultaneously were synergistic. Similar results have been obtained using highly enriched plasma membrane preparations.
#
i
J
400
E~ 300 ~'
J
o Control GTP o TRH
• TRH+GTP
20o ~
10o
c
E ~ 600 °~ ~ 500 o.~
400
300 200
,~...........
IOO
_._. -,X
~. ~ ' - ~ - o .. .. . . . . . . o .. .. . . . . . . . 1
3
5
,
IO
Time (rain)
FIG. 4. Timecourse of the effectsof TRH, GTP, and TRH plus GTP on InsPs and InsP2 accumulationin membranesuspensionsfrom GH3cells prelabeled with [3H]inos,_'tolto constant specificradioactivity.Reproduced with permissionfrom Straub and Gershengorn.2°
[ 10]
HORMONE-REGULATED PHOSPHOINOSIT1DE TURNOVER
| 11
Phosphoinositide Kinases (Steps 4 and 5 in Fig. 1) Final assay conditions 80 mM Tris-HCl, pH 7.9 1 mM EGTA 5 mM MgCI2 1 mM [3ep]ATP The reaction is initiated by the addition of 40-60 tzg of protein from a highly enriched plasma membrane preparation and the incubation is performed at 37°. Approximately 95% of the 32p label incorporated into lipids is accounted for by Ptdlns(4,5)P2 and Ptdlns4P; the remainder is present mainly as phosphatidic acid. Under these conditions, exogenous phosphoinositides do not serve as substrate. The kinase activities can also be detected as an increase in level of the polyphosphoinositides when plasma membranes isolated from [3H]inositol-prelabeled cells are used.
[10] H o r m o n e - R e g u l a t e d P h o s p h o i n o s i t i d e T u r n o v e r in P e r m e a b i l i z e d Cells a n d M e m b r a n e s
By T H O M A S
F. J. MARTIN
Introduction Agonist-regulated phosphoinositide hydrolysis as a receptor-associated transduction mechanism has been extensively studied in many cells as discussed elsewhere in this volume. Studies with intact cells, however, are insufficient for providing a detailed understanding of mechanisms. The recent development of permeabilized cells or membrane preparations for the study of receptor-regulated phosphoinositide hydrolysis has provided insights concerning the involvement of Ca 2+ and nucleotides (e.g., GTP) in the process. In addition, ceU-impermeant reagents (inhibitors or antibodies) can be utilized to probe mechanisms. Hormone-responsive membrane systems also offer the promise of biochemical reconstitution approaches by which an understanding of the relationship of receptors, phospholipid substrate, phosphodiesterase, and guanine nucleotide regulatory proteins can be achieved. The purpose of this chapter is to provide a summary of methods for studying hormone-regulated phosphoinositide turnover in permeable cells and in membranes. METHODS IN ENZYMOLOGY, VOL. 141
Copyright © 1987by Academic Press. Inc. All rights of reproduction in any form reserved.
INOSITOL PHOSPHOLIPIDS AND METABOLITES
[9]
[9] M e a s u r e m e n t o f L i p i d T u r n o v e r in R e s p o n s e to Thyrotropin-Releasing Hormone
By ATSUSHI IMAI and MARVIN C. GERSHENGORN Thyrotropin-releasing hormone (TRH), a tripeptide, binds specifically to receptors on the plasma membrane of anterior pituitary cells and rapidly stimulates the secretion of prolactin and thyrotropin (TSH). 1Because the mammalian anterior pituitary gland is composed of at least six cell types, only two of which are responsive to TRH and produce prolactin (mammotropes) or TSH (thyrotropes), the heterogeneity makes the cell population isolated from normal glands less than optimal for the study of the molecular mechanisms involved in TRH action. It is preferable to employ a homogeneous population of TRH-responsive cells so that the biochemical changes monitored upon stimulation by TRH can be presumed to occur uniformly in all cells of the type present. The most widely utilized homogeneous populations of pituitary mammotropes are GH cells, such as GH3 cells or GH4C1 cells. These are cloned cell lines derived from a rat pituitary tumor (MtT/W5) that produce prolactin and growth hormone. 2 The mechanism of TRH action in TSH-producing cells has not been as well studied as in mammotropic cells, at least partly because there is no cloned TRH-responsive, TSH-producing cell line. In many cells, including these TRH-responsive pituitary cells, the interaction of stimuli with cell surface receptors enhances the turnover of membrane phospholipids. It has been proposed that the increased metabolism of phosphoinositides constitutes a signal-transducing mechanism that is initiated by stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2], a minor but metabolically labile polyphosphoinositide, by a phospholipase C to generate two putative intracellular messengers, water-insoluble 1,2-diacylglycerol (1,2-DG), and watersoluble inositol trisphosphate (InsP3). 1,2-DG, which appears to serve as an intracellular mediator by activating protein kinase C, and InsP3, which acts by releasing Ca 2÷ from a nonmitochondrial store, may function in a coordinated or synergistic manner to activate a wide variety of cellular processes. This section describes the methodology that we have employed to study TRH stimulation ofphosphoinositide metabolism in intact GH3 cells and in membranes isolated from GH3 cells. The observations J M. C. Gershengorn, Annu. Rev. Physiol. 48, 515 (1986). 2 p. M. Hinkle and A. H. Tashjian, Jr., in "Biochemical Actions of Hormones" (G. Litwack, ed.), p. 269. Academic Press, New York, 1977.
METHODS IN ENZYMOLOGY, VOL. 141
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
[9l
LIPID TURNOVER IN RESPONSE TO T R H
101
made using a highly enriched preparation of plasma membranes appear to constitute the most convincing evidence that turnover of plasma membrane phosphoinositides is primarily stimulated during cell activation by TRH. Preparation of Radiolabeled GH3 Cells
Reagents GH3 cells (supplied by American Type Tissue Collection) Ham's F10 medium (supplied by GIBCO); supplemented with 15% horse serum and 2.5% fetal bovine serum Balanced salt solution (BSS): 135 mM NaC1, 4.5 mM KC1, 1.5 mM CaC12, 0.5 mM MgCI2, 5.6 mM glucose, and 10 mM HEPES, pH 7.4 0.02% EDTA in phosphate-buffered isotonic saline Procedure. GH3 cells are grown as monolayer cultures in 150-175 cm 2 flasks in Ham's FI0 medium supplemented with 15% horse serum and 2.5% fetal bovine serum under a humidified atmosphere of 5% CO2 and 95% air at 37°. The phospholipids can be labeled by incubating the cells in the presence of a variety of radioactive precursors including phosphate, glycerol, inositol, or various fatty acids. Cells may be labeled with any of these precursors so that the radioactivity incorporated into lipids achieves a constant specific activity or for brief periods so that the specific radioactivity in the lipids is changing. The ability to label GH3 cell lipids to constant specific activity is a great advantage of this cell system because the changes in the level of the radioactivity in a given molecular species then directly reflects changes in its mass. Prelabeling GH3 cells to a constant specific radioactivity is possible because these cells are a permanent cell line and can be grown in medium supplemented with one or more of these radiolabeled precursors for 48 hr, a time that is sufficient for the radiolabel in all major lipids to attain constant specific activity. 3,4 One microCurie of [3H]inositol, 10/zCi of [32p]phosphate, and, for example, 0.1/xCi of [3H]arachidonic acid or 0.2/xCi of [14C]stearic acid are usually added per milliliter of medium. Because [3H]inositol specifically labels the phosphoinositides, the majority of the more recent studies of the effects of TRH in GH3 cells have utilized [3H]inositol-prelabeled cells. Under these conditions, over 95% of 3H radioactivity in the lipid fraction is found in phosphatidylinositol (Ptdlns), lysophosphatidylinositol, phosphatidylinositol 4-monophosphate (PtdIns4P), and PtdIns(4,5)Pz ; there is approximately 4.5 × 103 cpm/nmol Ptdlns. After labeling, the cells are harvested 3 M. J. Rebecchi, R. N. Kolesnick, and M. C. Gershengorn, J. Biol. Chem. 258, 227 (1983). 4 M. J. Rebecchi and M. C. Gershengorn, Biochem. J. 216, 287 (1983).
102
INOSlTOL PHOSPHOLIPIDS AND METABOLITES
19]
by incubating them in 0.02% EDTA in phosphate-buffered saline for 5-10 min. The cells are then collected by centrifugation at 180 g for 5 min, resuspended in BSS (1-5 x 106 cells/0.1 ml), and allowed to incubate at 26° for 20-30 min. We study the effects of TRH on the metabolism of the phosphoinositides in GH3 cells exclusively in cells in suspension; however, other workers have utilized cells in monolayer culture and have obtained similar results. The majority of experiments with GH3 cells prelabeled under conditions such that specific radioactivity of the lipids is changing are conducted with cells labeled with [32P]phosphate: The major advantage of this experimental design is that even small changes in the metabolism of all phospholipids can be detected as these are magnified by the increasing specific radioactivity of the phosphate being incorporated. For these experiments, GH3 cells are harvested as described above and incubated in BSS (1-5 × 106 cells/0.1 ml) containing 50-100/zCi of [32P]phosphate/0.1 ml at 26 ° for 40-60 min. [32P]Phosphate in the lipid fraction is recovered mainly in the more labile phospholipids whose final synthetic step is catalyzed by a kinase, in particular, Ptdlns(4,5)P2, Ptdlns4P, and phosphatidic acid. The experimental incubations of cells prelabeled to constant specific radioactivity and of cells prelabeled briefly with [32P]phosphate are performed identically. Cells are separated from the preincubation medium by centrifugation at 180 g for 5 min, washed, and resuspended in fresh BSS without radioactive precursor (1-5 x 106 cells/0.1 ml). Portions of the cell suspension (0.09 ml) are placed in individual test tubes and 0.01 ml TRH (final concentration, 0.01 to 1000 nM) or vehicle is added. The incubation is terminated by adding 1.0 ml chloroform/methanol/concentrated HCI (I00: 100: 1, v/v/v). In some experiments with cells prelabeled with [3H]inositol, the experimental incubation is performed in BSS supplemented with 10 m M LiC1; LiCI is used to inhibit the enzyme inositol monophosphate (InsP) phosphatase that converts InsP to inositol and thereby allow for the increase in inositol phosphates caused by TRH to be more readily measured. Lipid Extraction and Analysis
Reagents Chloroform/methanol/concentrated HC1 (100: 100: 1, v/v/v) 10 m M EDTA 5 M. J. Rebecchi, M. E. Monaco, and M. C. Gershengorn, Biochem. Biophys. Res. Cornmun. 101, 124 (1981).
[9]
LIPID TURNOVER IN RESPONSE TO T R H
103
Preequilibrated upper phase: Upper phase of chloroform/methanol/ concentrated HCI/HzO/lO mM EDTA (50 : 50 : 0.5 : 10 : 2.5, v/v/v/v/v) Preequilibrated lower phase: Lower phase of above mixture Silica gel H plates Silica gel G plates Procedure. Lipids are extracted according either to the methods of Bligh and Dyer 6 or of Foich. v Acidification of the chloroform/methanol solutions and EDTA permit better recovery of polyphosphoinositides from the cells; the concentration of HC1 used does not cause hydrolysis of phospholipids. The chloroform/methanol/HC1 extract is separated into two phases by adding 0.25 ml of 10 m M EDTA. After brief centrifugation, the lower phase is removed. Two-tenths milliliter preequilibrated lower phase is used to wash the upper phase to remove residual lipids. The lower phases are combined and washed with 1 ml preequilibrated upper phase at 0-4 ° in order to remove any residual water-soluble material. The resulting chloroform phases are dried under a stream of N2 and immediately redissolved in a small volume of chloroform/methanol (1:1, v/v). Portions of the samples are applied under N2 to silica gel plates. No single thin-layer chromatographic system adequately resolves all the most important lipids. We have, therefore, used a series of different solvent systems and chromatographic plates to isolate and measure the lipids of interest. Major phospholipids are separated on silica gel H plates developed in one dimension with chloroform/methanol/acetic acid/H20 ( 5 0 : 3 0 : 8 : 4 , v/v/v/v). 8 This method is usually employed for screening changes in phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, and Ptdlns (Ptdlns is not separated from phosphatidylserine), especially in experiments in which cells are labeled with [32p]phosphate. Ptdlns can be measured using this system when the cells are labeled specifically with [3H]inositol. We use silica gel G plates developed in one dimension with chloroform/pyridine/70% formic acid (50 : 30 : 7, v/v/v) to more adequately isolate phosphatidic acid and phosphatidylethanolamine. 9 1,2-Diacylglycerol (I,2-DG), the lipid product of phosphoinositide degradation due to phospholipase C activation, is isolated from other lipid compounds either on silica gel G plates using the upper phase of ethyl acetate/2,2,4-trimethylpentane/acetic acid/H20 (90:50 : 20 : 100, v/v/v/v) ~°or on silica gel G plates impregnated with 0.4 M boric acid with a E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 7 j. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem. 226, 497 (1957). 8 V. P. Skipski, R. A. Peterson, and M. Barclay, Biochem. J. 90, 374 (1964). 9 R. V. Farese, A. M. Sabir, and R. E. Larson, J. Biol. Chem. 255, 7232 (1980). to E. G. Lapetina and P. Cuatrecasas, Biochim. Biophys. Acta 573, 394 (1979).
104
INOSITOL PHOSPHOLIPIDS AND METABOL1TES ATP
f/
\
ADP
= PtdIns.,
ATP
~ PtdIns4P 7
~
I0
InsP +
DG
C TP ~./Z'J
CDP'DG'IV
~_
~ PtdIns(4,5)P
l
9
I n s P 2 "I + DG
J PPi.
ADP
z
G
3 Inositol
[9]
1'
8
InsP3 +
DG
)
, L v PtdA
FIG. 1. Metabolic pathways ofphosphoinositides. Enzymes: (1) Ptdlns(4,5)P2 phospholipase C; (2) Ptdlns4P phospholipase C; (3) Ptdlns phospholipase C; (4) Ptdlns kinase; (5) Ptdlns4P kinase; (6) Ptdlns(4,5)P2 phosphomonoesterase; (7) Ptdlns4P phosphomonoesterase; (8) InsP3 phosphomonoesterase; (9) InsP2 phosphomonoesterase; (10) InsP phosphatase; (11) 1,2-DG kinase; (12) PA:CTP cytidyltransferase; (13) CDP-DG (CMP-PA):inositol 3-phosphatidyl transferase. Reproduced with permission from Rebecchi and Gershengorn.13~
solvent system consisting of chloroform/acetone (96:4, v/v). 11 Both of these systems yield adequate separation also of triacylglycerol, 1,3-diacylglycerol, 1-monoacylglycerol, 2-monoacylglycerol, and free fatty acids, such as arachidonic acid from phospholipids. Two-dimensional separations are necessary to resolve the majority of the phospholipids including the lysophospholipids, the products of the hydrolysis by a phospholipase A2 enzyme. There are several useful two-dimensional chromatographic systems. For example, the thin-layer plates can be either 2.5% magnesium acetate-impregnated HPTLC or silica gel 60 that are developed in the first dimension with chloroform/methanol/13.5 N NH4OH (65 : 35 : 5.5, v/v/v) and with chloroform/methanol/acetone/acetic acid/H20 (3 : 1 : 4 : 1 : 0.5, v/v/v/v/v) in the second dimension) z,13 The phospholipid pathway primarily affected by T R H in GH3 cells is the polyphosphoinositide cycle (see Fig. 1). 13a The effects of TRH on these lipids and inositol sugars are best studied in cells labeled to constant H y. Kameyama, S. Yoshioka, and Y. Nozawa, Biochirn. Biophys. Acta 665, 195 (1981). 12M. J. Broekman, J. W. Ward, and A. J. Marcus, J. Clin. lnvest, 66, 275 (1980). 13A. Imai, Y. Ishizuka, S. Nakashima, and Y. Nozawa, Arch. Biochem. Biophys. 232, 259 (1984). ~3aM. J. Rebecchi and M. C. Gershengorn, in "The Receptors" (P. M. Conn, ed.), Vol. 3, p. 173. Academic Press, New York, 1985.
[9]
LIPID TURNOVER IN RESPONSE TO T R H
1 TRH
I
I
I
~
105
I
100
~
_
,r.
8O
-o 2 E o o 60
:
40
DPI I
2O I
I
0
1
I
I
2 3 4 Time (minutes)
I
I
5
FIG. 2. Time course of the changes in the levels of the phosphoinositides induced by TRH in GH3 cells prelabeled with [3H]inositol to constant specific radioactivity. TRH (1 /~M) stimulates rapid loss by 15 sec of Ptdlns(4,5)P2 and Ptdlns4P. Initial levels of Ptdlns(4,5)P~, Ptdlns4P, and Ptdlns was 100, 150, and 5000 cpm/106 ceils, respectively. Reproduced with permission from Rebecchi and Gershengorn. 4
specific radioactivity with [3H]inositol as described above. The phosphoinositides are resolved well by sequential ascending chromatography on silica gel H plates in chloroform/methanol/4 N NH4OH (45 : 35 : 10, v/v/v) containing 2 mM cyclohexylenedinitrilotetraacetic acid (CDTA) followed by chloroform/methanol/acetic acid/H20 (50 : 30 : 8 : 4, v/v/v/v): Development with both solvent systems in the same dimension and the calcium chelator CDTA permit the highly charged polyphosphoinositides, PtdIns4P and PtdIns(4,5)P2, to migrate from the origin. The Rr values for PtdIns(4,5)P2, PtdIns4P, lysoPtdIns, and Ptdlns are 0.11, 0.29, 0.68, and 0.92, respectively. Figure 2 illustrates the results of experiments in which the effects of TRH on the phosphoinositides were measured in GH3 cells prelabeled to constant specific radioactivity with [3H]inositol.4 The rapid effects of TRH to decrease the levels of PtdIns(4,5)P2 and PtdIns4P, and more slowly of PtdIns, are apparent. Alternatively, the compounds are separated on high-performance thin-layer chromatography (HPTLC) plates impregnated with 1% potassium oxalate developed with chloroform/acetone/methanol/acetic acid/H20 (40:15 : 13 : 12 : 8, v/v/v/v/v). 14 When the fatty acid residues of the phosphoinositides are not of interest, another method is to deacylate the phosphoinositides by incubating the 14 j. Jolles, H. Zwiers, A. Dekker, K. W. A. Wirtz, and W. H. Grispen, Biochem. J. 194, 283 (1981).
106
INOSITOL PHOSPHOLIPIDS AND METABOLITES
[9]
lipid with methanolic NaOH (0.2 N) for 15 min ~5,~6and separate the resulting water-soluble glycerophosphoinositol, glycerophosphoinositol 4monophosphate, and glycerophosphoinositol 4,5-bisphosphate on small anion-exchange columns (see the section on inositol phosphate analysis). Major phospholipids and neutral lipids can be visualized by staining with iodine vapor. In GH3 cells, as in most cells, the content of polyphosphoinositides is insufficient for detection in this way unless carrier standards are added during chromatography. Another method to detect phospholipids is by autoradiography when the cells are labeled with [32p]phosphate (or by adding 32p-labeled standards). We expose the thin-layer plates to Kodak X-Omat films in their covers for 16-48 hr. This is an especially useful method to detect the endogenous polyphosphoinositides from cells that are labeled briefly with [32p]phosphate because these highly labile phospholipids incorporate a major fraction of the radiolabel in lipids under these conditions. Spots are scraped into vials and radioactivity is determined. The phospholipid phosphate is determined by measuring the phosphorus content of acid hydrolysates of chromatogram scrapings using, for example, the method of Chen et al. J7 As little as 2 nmol phosphorus can be detected by this procedure. Analysis of Inositol Phosphates Reagents
0.1 M formic acid 0.2, 0.4, and 0.7 M ammonium formate in 0.1 M formic acid AG l-X2 resin (acetate form, 200-400 mesh): Supplied by Bio-Rad 5 m M myo-inositol in 0.1 M formic acid The inositol phosphates--inositol monophosphate (InsP), inositol bisphosphate (InsP2), and inositol trisphosphate (InsP3)--are formed either directly by the phospholipase C-mediated hydrolysis of the phosphoinositides and/or by sequential dephosphorylation from InsP3 ; InsP3 is the only unique reaction product as it can be formed only from the hydrolysis of Ptdlns(4,5)P2 by a phospholipase C. The inositol phosphates and inositol can be isolated by anion exchange chromatography, electrophoresis, paper chromatography, or high-pressure liquid chromatography (HPLC). The most widely used, simple technique is chromatography, using small anion-exchange resin columns. Because anion exchange chromatography 15 R. B. Ellis, T. Galliard, and J. N. Hawthorne, Biochem. J. 88, 125 (1963). 16 R. M. C. Dawson, N. Hemington, and J. B. Davenport, Biochem. J. 84, 497 (1962). ~7p. S. Chen, Jr., T. Y. Toribora, and H, Warner, Anal. Chem. 28, 1756 (1956).
[9]
LIPID TURNOVER IN RESPONSE TO T R H
I TRH 500i
i
i
i
107
i
• [3H]l~0sit0t 1,4,5-ltiph0sphate 1
Z~ [3H]Inositol 1,4-diphosoh6te
I I
I • [3H]InOSifOJ 1" rnonophosphote/
-~ 4oo o Q) o
300 2O0 100 1"
t
~ _
J_
t
/
2 5 4 5 Time (minutes) FIG. 3. Time course of the changes in the levels of the inositol phosphates induced by TRH in GH3 cells prelabeled with [3H]inosiIol to constant specific radioactivity. TRH (1 /~M) stimulates rapid and transient increase in InsP3 and InsP2 and a slower increase in InsP. Initial levels of InsP3, InsP2, InsP, and inositol were 150,500, 2000, and 10,000 cpm/5 x 106 cells, respectively. Reproduced with permission from Rebecchi and Gershengorn. 4 0
~
does not isolate the inositol phosphates from all other small, water-soluble molecules, this analysis is primarily applied to measurements of the inositol sugars from cells prelabeled with [3H]inositol. Procedure. The water-methanol upper phase of the cell extract contains the water-soluble inositol phosphates and inositol. After removal of the interface and lower phase, the upper phase is dried under a stream of N2 and the residue is redissolved in 0.1-0.2 ml of 0.1 M formic acid. The samples are then loaded onto freshly prepared 1-ml columns of resin (AG I-X2), which is, prior to use, swollen in 0.1 M formic acid containing 5 m M myo-inositol. After the sample is applied, the column is washed with 10 to 20 ml of 0.1 M formic acid to remove inositol, which is not retained by the resin and thus appears in the column flow-through, before eluting the inositol phosphates. This wash step is important because there is a much greater amount of 3H radioactivity in inositol in these cells than in the inositol phosphates. The inositol phosphates are eluted sequentially by using 0.2 M ammonium formate/0.1 M formic acid (for InsP), 0.4 M ammonium formate/0.1 M formic acid (for InsP2), and 0.7 M ammonium formate/0.1 M formic acid (for InsP3). TMIn routine assays, 10 fractions (1 ml each) are collected for each eluent. The 3H radioactivity is measured in each fraction and the content of each inositol phosphate is calculated from the counts in each peak. Figure 3 illustrates the results of experiments, 18 C. P. Downes and R. H. Michell, Biochem. J. 198, 133 (1981).
108--
INOSITOL PHOSPHOLIPIDS AND METABOLITES
[9]
from which the lipid analyses were presented in Fig. 2, of the effects of TRH on the levels of the inositol phosphates and inositol in GH3 cells prelabeled to constant specific radioactivity with [3H]inositol. 4 TRH caused a rapid increase in the levels of InsP3 and InsP2 that was followed by a slower rise in the level of InsP. It is important to note again that this system does not isolate the inositol phosphates from other small, watersoluble molecules, such as nucleotides and free phosphate, and, therefore, improved techniques are needed. New methods would be especially useful for analysis of cells that are labeled with [32P]phosphate that would permit more detailed kinetic studies of the interconversions of the inositol phosphates.
Measurement of Phosphoinositide Phospholipases C and Kinases Associated with the Plasma Membrane
In GH3 cells, TRH causes a rapid decrease in the level of Ptdlns(4,5)P2 and Ptdlns4P (Fig. 2) and stimulates a marked, transient increase in InsP3, the unique product of phospholipase C-mediated hydrolysis of Ptdlns(4,5)P2, and in InsP2 (Fig. 3). These findings prove that TRH stimulates the phospholipase C-mediated hydrolysis of Ptdlns(4,5)P2. It is unclear whether there is direct hydrolysis of Ptdlns4P that m a y also lead to the formation of InsP2. These phenomena seem to occur in the plasma membrane. Thus, we have studied the enzyme activities catalyzing these events in preparations of membranes isolated from GH3 cells.
Preparations of Membrane Fractions from GHs Cells Reagents Lysis buffers: (A) 2 m M MgC12, 25 m M Tris-HC1, pH 6.9 (B) 0.5 m M dithiothreitol (DTT), 1 mM EGTA, 1 mM sodium bicarbonate, 10 m M HEPES, pH 7.9 30, 36, 41, 45, and 50% (w/v) sucrose in lysis buffer B Procedure. The method to prepare a crude membrane fraction from GH3 cells is essentially that described by Hinkle and Phillips.19 Cells are suspended in a volume of lysis buffer A equal to four to five times the volume of the cell pellet at 0°. After 10 min, the suspension is homogenized with a Teflon-glass homogenizer and centrifuged at 400 g for 5 min. 19p. M. Hinkle and W. J. Phillips, Proc. Natl. Acad. Sci. U.S.A. 81, 6183 (1984).
[9]
LIPID TURNOVER IN RESPONSE TO T R H
109
The supernatant is centrifuged at 7500 g for 10 min and the pellet is resuspended in assay buffer at a protein concentration of 2-3 mg/ml. 2° To prepare a highly enriched fraction of plasma membranes, the cells are suspended in a volume of lysis buffer B equal to four to five times the volume of the cell pellet. The cells are lysed by incubating them at 37° for 4.5 rain, followed by addition of 15 vol of ice-cold lysis buffer and rapid mixing on a vortex mixer for 0.5 rain at 0-4 °. The homogenate is centrifuged at 800 g for l0 rain to remove nuclei and cell debris. The supernatant is centrifuged again at 100,000 g for l hr. The resulting pellet is resuspended in 4 ml of lysis buffer B that is then layered on top of a discontinuous sucrose density gradient consisting of 30, 36, 41, 45, and 50% sucrose in lysis buffer B and centrifuged at 100,000 g for 1 hr. Five narrow bands are obtained at the interfaces. The upper two bands are collected as the plasma membrane fraction, diluted with lysis buffer, and centrifuged at 100,000 g for 1 hr. (The lower two bands are enriched in endoplasmic reticulum.) The final pellet is resuspended in lysis buffer B and used immediately or stored at - 7 0 °. The purity of the preparations can be assessed by the relative recoveries of marker enzymes. We commonly use Na +,K+-ATPase as a marker for plasma membranes, succinate dehydrogenase for mitochondria, and NADH dehydrogenase for endoplasmic reticulum. The plasma membrane fraction is enriched by approximately 20-fold in Na+,K+-ATPase activity.
Assays of Membrane-Associated Enzymes Polyphosphoinositide-Specific Phospholipase C (Steps I and 2 in Fig. 1) Final assay conditions 25 mM Tris-HC1, pH 6.9 1 mM Na2EDTA 2 mM ATP 6 mM MgCI: 2 mM dithiothreitol 1 mM ouabain 10 mM LiCI (to inhibit the conversion of InsP to inositol) The reaction is initiated by the addition of 40-60/zg membrane protein isolated from GHa cells that have been prelabeled with pH]inositol to constant specific radioactivity. Factors to be tested, such as TRH and z0 R. E. Straub and M. C. Gershengorn, J. Biol. Chem. 261, 2712 (1986).
1I0
[9]
1 N O S I T O L PHOSPHOLIPIDS A N D M E T A B O L I T E S
guanine nucleotides, are added to the buffer and the incubation is performed at 37°. The reaction is terminated by addition of chloroform/methanol/HCl, and phospholipid and inositol phosphate extractions and measurements are performed as outlined above. InsP3, InsP:, and InsP are produced. As in studies with intact cells, the observations made with this in vitro experimental design do not allow for a distinction as to whether InsP2 and InsP are formed from direct phospholipase C-mediated hydrolysis of Ptdlns4P and Ptdlns, respectively, or from dephosphorylation from InsP3. Figure 4 illustrates the results of an experiment that measured the effects of TRH and GTP on the formation of InsP3 and lnsP2.20 TRH and GTP caused rapid increases in the content of both inositol polyphosphates. The level of InsP3 decreased with time, suggesting the presence of an InsPs degrading enzyme(s). The effects of TRH and GTP when added simultaneously were synergistic. Similar results have been obtained using highly enriched plasma membrane preparations.
#
i
J
400
E~ 300 ~'
J
o Control GTP o TRH
• TRH+GTP
20o ~
10o
c
E ~ 600 °~ ~ 500 o.~
400
300 200
,~...........
IOO
_._. -,X
~. ~ ' - ~ - o .. .. . . . . . . o .. .. . . . . . . . 1
3
5
,
IO
Time (rain)
FIG. 4. Timecourse of the effectsof TRH, GTP, and TRH plus GTP on InsPs and InsP2 accumulationin membranesuspensionsfrom GH3cells prelabeled with [3H]inos,_'tolto constant specificradioactivity.Reproduced with permissionfrom Straub and Gershengorn.2°
[ 10]
HORMONE-REGULATED PHOSPHOINOSIT1DE TURNOVER
| 11
Phosphoinositide Kinases (Steps 4 and 5 in Fig. 1) Final assay conditions 80 mM Tris-HCl, pH 7.9 1 mM EGTA 5 mM MgCI2 1 mM [3ep]ATP The reaction is initiated by the addition of 40-60 tzg of protein from a highly enriched plasma membrane preparation and the incubation is performed at 37°. Approximately 95% of the 32p label incorporated into lipids is accounted for by Ptdlns(4,5)P2 and Ptdlns4P; the remainder is present mainly as phosphatidic acid. Under these conditions, exogenous phosphoinositides do not serve as substrate. The kinase activities can also be detected as an increase in level of the polyphosphoinositides when plasma membranes isolated from [3H]inositol-prelabeled cells are used.
[10] H o r m o n e - R e g u l a t e d P h o s p h o i n o s i t i d e T u r n o v e r in P e r m e a b i l i z e d Cells a n d M e m b r a n e s
By T H O M A S
F. J. MARTIN
Introduction Agonist-regulated phosphoinositide hydrolysis as a receptor-associated transduction mechanism has been extensively studied in many cells as discussed elsewhere in this volume. Studies with intact cells, however, are insufficient for providing a detailed understanding of mechanisms. The recent development of permeabilized cells or membrane preparations for the study of receptor-regulated phosphoinositide hydrolysis has provided insights concerning the involvement of Ca 2+ and nucleotides (e.g., GTP) in the process. In addition, ceU-impermeant reagents (inhibitors or antibodies) can be utilized to probe mechanisms. Hormone-responsive membrane systems also offer the promise of biochemical reconstitution approaches by which an understanding of the relationship of receptors, phospholipid substrate, phosphodiesterase, and guanine nucleotide regulatory proteins can be achieved. The purpose of this chapter is to provide a summary of methods for studying hormone-regulated phosphoinositide turnover in permeable cells and in membranes. METHODS IN ENZYMOLOGY, VOL. 141
Copyright © 1987by Academic Press. Inc. All rights of reproduction in any form reserved.