Journal of Neurochemistry Raven Press. Ltd., New York 0 1990 International Society for Neurochemistry

Production of 1,2-Diacylglycerol in PC 12 Cells by Nerve Growth Factor and Basic Fibroblast Growth Factor Joseph G. Altin and Ralph A. Bradshaw Department Qf‘Biological Chemistry, CaliJ&niu Coflcge of Medicine, University of California, Irvine, t ‘alifornia, U.S.A.

Abstract: The addition of nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) to PC12 cells prelabeled with [3H]inositol and preincubated for 15 min in the presence of 10 mA4 LiCl stimulated the production of inositol phosphates with maximal increases of 120- 180% in inositol monophosphate (IP), 130-200% in inositol bisphosphate (IP2), and 4550% in inositol trisphosphate (IP,) within 30 min. The majority of the overall increase (approximately 85%) was in IP; the remainder was recovered as IP2 and IP3 (approximately 10% as IP2 and 5% as IP3). Under similar conditions, carbachol (0.5 m M ) stimulated about a l0-fold increase in IP. a sixfold increase in IP2, and a fourfold increase in IP3. The mass level of 1,2diacylglycerol (DG) in PC 12 cells was found to be dependent on the incubation conditions; in growth medium [Dulbecco’s modified Eagle’s medium (DME) plus serum], it was around 6.2 rnol %, in DME without serum, 2.5 mol %, and after a 15-min incubation in Dulbecco’s phosphate-buffered saline, 0.62 mol %. The addition of NGF

and bFGF induced a n increase in the mass level of D G of about twofold within 1-2 min, often rising to two- to threcfold by 15 min, and then decreasing slightly by 30 min. This increase was dependent on the presence of extracellular Ca2+, and was inhibited by both phenylarsinc oxide (25 ~ L Mand ) 5’-deoxy-5‘-methylthioadenosine(3 mM). Under similar conditions, 0.5 m M carbachol stimulated the production of DG to the same extent as 200 ng/ml NGF and 50 ng/ml bFGF. Because carbachol is much more effective in stimulating the production of inositol phosphates, the results suggest that both N G F and bFGF stimulate the production of D G primarily from phospholipids other than the phosphoinositides. Key Words: PC I2 cells- 1,2-Diacylglycerol-Nerve growth factor-Inositol phosphates. Altin J. G . and Bradshaw R. A. Production of 1,2-diacylglycerol in PC 12 cells by nerve growth factor and basic fibroblast growth factor. J. Nezuochem. 54, 1666- 1676 ( 1990).

The mechanism by which polypeptide growth factors like nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) promote either differentiation or proliferation in responsive cells is not yet fully understood. Both of these factors stimulate the rat pheochromocytoma cell line PC 12, which has been widely used to study neuronal differentiation. In culture, PC 12 cells grow as round chromaffin-like cells undergoing cell division every 48-72 h (Greene and Tischler, 1982). On addition of NGF (or bFGF), the cells stop dividing, extend numerous processes, and display morphological changes characteristic of differentiated neurons (Greene and Tischler, 1982). This process is accompanied by changes in ion fluxes (Boonstra el al., 1983; Morgan and Curran, 1986; Pandiella-Alonso et al., 1986), by phosphorylation of specific proteins (Landreth and

Williams, 1987), by alteration in the activity of numerous enzymes including ornithine decarboxylase (Greene and McGuire, 1978), by cyclic AMP- and Ca’+/phospholipid-dependent protein kinases (Blenis and Erikson, 1986; Cremins et al., 1986; Hama et al., 1986, 1987; Rowland et al., 1987), and by induction of specific oncogenes, such as c-fus (Curran and Morgan, 1985; Greenberg et al., 1985; Kruijer et al., 1985; Milbrandt, 1986) and a variety of other genes (Greene and Rein, 1977; Greene and Tischler, 1982; Kujubu et al., 1987; Leonard et al., 1987). Among the molecular events proposed to mediate these changes are the protein p2lras (Bar-Sagi and Feramisco, 1985; Hagag et al., 1986; Yu et al., 1988) and the activation of protein kinase C (Hall et al., 1988). The addition of NGF to PC 12 cells stimulates the in-

Received July 3, 1989; revised manuscript received September 18, 1989; accepted October 4, 1989. Address correspondence and reprint requests to Dr. R. A. Bradshaw at Department of Biological Chemistry, California College of Medicine, University of California, Irvine, CA 927 17, U.S.A. Abbreviations used: bFGF, basic fibroblast growth factor; DG, I ,2-

diacylglycerol; DME, Dulbecco’s modified Eagle’s medium; IP, inositol monophosphate; IP, , inositol bisphosphate; IP, , inositol trisphosphate; IP4, inositol tetrakisphosphate; MTA, 5’-deoxy-5‘-methylthioadenosine; NGF, nerve growth factor; PAO, phenylarsine oxide; PBS, phosphate-bufferedsaline.

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1.2-DIACYLGLYCEROL PRODUCTION BY NGF AND bFGF corporation of [32P]04 into phosphatidylinositol, phosphatidic acid, and, to a lesser extent, other phospholipids such as phosphatidylcholine (Traynor et al., 1982). In addition, NGF has been found to stimulate a rapid production of inositol phosphates in these cells (Contreras and Guroff, 1987). The rapid alteration in the metabolism of inositol phospholipids following the addition of NGF, and the stimulation of neurite outgrowth in PC 12 cells by an increased concentration of exogenous KCl (Schubertet al., 1978), which was found to stimulate the turnover of phosphatidylinositol (Traynor, 1984), have led to the suggestion that the stimulation of phosphoinositide hydrolysis by NGF is an early event in the signal transduction cascade leading to differentiation and the induction of neurite outgrowth (Traynor, 1984; Contreras and Guroff, 1987). Also consistent with phosphoinositide hydrolysis as a signaling mechanism of NGF are the observations that protein kinase C activity increases three- to fourfold within the first hour of exposing PC12 cells to NGF (Hama et al., 1986, 1987). Moreover, phorbol myristate acetate (an activator of protein kinase C) induces neurite outgrowth in CNS neurons (Moskal and Momson, f987), and sphingosine(a reversible inhibitor of protein kinase C) blocks NGF-induced neurite outgrowth in PC12 cells (Hall et al., 1988). In the absence of detailed studies on the effects of NGF on 1,2-diacylglycerol (DG) production (a physiological activator of protein kinase C), the mechanism by which NGF increases protein kinase C activity in this system is uncertain. Moreover, bFGF, a mitogen in certain cell types, has been shown to act as a neurotrophic agent (Morrison et al., 1986; Walicke et al., 1986) and to induce the same spectrum of responses as NGF in PC I2 cells (Rydel and Greene, 1987; Koizumi et al., 1988). However, bFGF is generally not considered to act by stimulating phosphoinositide hydrolysis in other systems (Magnaldo et al., 1986; Chambard et al., 1987); thus, the question arises as to the role of phosphoinositide hydrolysis in the induction of neurite outgrowth by NGF, and whether NGF and bFGF might elicit neurite outgrowth by distinct signal transduction pathways. In this report, the effects of NGF and bFGF on the production of inositol phosphates and DG in PC I2 cells in culture have been examined. The results suggest that increases in inositol phosphates induced by NGF and bFGF are small compared with increases induced by the muscarinic agonist carbachol, and that the increases in DG induced by both growth factors are greater than can be accounted for by the hydrolysis of the phosphoinositides alone.

EXPERIMENTAL PROCEDURES Materials PC12 cells were obtained from Dr. David Schubert, Salk Institute, San Diego, CA, U.S.A. Dulbecco’s modified Eagle’s medium (DME) and DME without inositol were obtained

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from Flow Laboratories. Fetal calf serum and heat-inactivated horse serum were obtained from Irvine Scientific (Santa Ana, CA, U.S.A.) and Cell Culture Laboratories (Cleveland, OH, U.S.A.), respectively. B-NGF was prepared according to the method of Mobley et al. (1976). A bFGF analog in which all half-cystine residues were replaced by serines was used for all experiments with bFGF. This analog, which was kindly provided by Dr. Gary M. Fox, Amgen Inc., Thousand OaksCA, U.S.A., has been reported to be as active as, but more stable than, the recombinant forms of bovine and human bFGF with the native sequence (Fox et al., 1988). In these experiments, the effect of the bFGF analog on the production of inositol phosphates and DG in PC12 cells was found to be indistinguishable from tha1 of the recombinant native bovine bFGF (data not shown). 1,2-Diolein standard (1,2-dioleoyl-ruc-glycerol),phenylarsine oxide (PA0), and 5’-deoxy5’-methylthioadenosine (MTA) were obtained from Sigma. Chloroform, methanol, HPLC-grade hexane, isopropanol, and acetic acid were obtained from Fisher Scientific. myo[3H]Inositol was obtained from DuPont NEN Products, and anion exchange resin AG-1 X 8 was from Bio-Rad. Other reagents were of analytical grade.

Cell culture PC 12 cells were adapted to grow in DME supplemented with 10% fetal calf serum, 5% horse serum, 100 U/ml of penicillin, 100 pg/ml of streptomycin, and 0.25 pg/ml of fungizone. Stock PC12 cells were grown in this medium in Belco tissue culture flasks (T75 or T150) essentially as described (Greene and Tischler, 1982). For each experiment, the cells were subcultured by seeding at a density of approximately 2 X lo4 cells/cm2 and then grown in this medium until 50-70% confluent. All cells were maintained at 37°C in a humidified atmosphere of 5% COz; they were fed every 2 days and passed weekly.

Measurement of inositol phosphates For measurement of changes in inositol phosphates, stock PC 12 cells were seeded and grown in Belco T75 flasks, washed three times with warmed Dulbecco’s phosphate-buffered saline (PBS) containing 137 mMNaC1, 2.7 mMKC1, 0.5 mM MgClz, 1.5 mM KH2P04, 8.06 mM NaH2P04,0.9 mM CaClz, and 10 mMglucose (pH 7.4), and then incubated for 40-48 h in DME medium without inositol but containing 0.4 pCi/ml my~-[~H]inositol and supplemented with 5% fetal calf serum and 2.5% heat-inactivated horse serum. To increase cell labeling efficiency, the fetal calf serum and horse serum used in these incubations were charcoal-stripped to remove unlabeled inositol. At the end of this incubation period, the cells were washed three times with warmed PBS, and then preincubated at 37OC in this medium with or without 10 mM LiCl for 15 min before the addition of growth factors. NGF and bFGF (dissolved in PBS) were added directly to the flasks, and after a suitable incubation period the medium was quickly removed and 15%trichloroacetic acid was added to stop the reaction. The cells were scraped into this trichloroacetic acid and transferred to a plastic tube, then centrifuged for 2-3 min to pellet the precipitate. The trichloroacetic acidsoluble extract (supernatant) was transferred into a 15-ml glass tube and then extracted four times with 4 ml of ether to remove the trichloroacetic acid. Separation of the inositol phosphates from this extract was camed out by ion-exchange chromatography using small columns containing 0.6 ml of ion exchange resin AG-1 X 8. The samples were neutralized with HEPES/NaOH and added to the columns. After a first

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J. G. ALTIN AND R. A. BRADSHAW

wash with 8 ml of a solution containing 60 m M sodium formate and 5 mM disodium tetraborate, the inositol phosphates were eluted with 2 X 2.5 ml of different concentrations of ammonium fomate [0.2 M for inositol monophosphate (IP); 0.4 M for inositol bisphosphate (IP2);0.8 M for inositol trisphosphate (IP3), and 1.2 M for inositol tetrakisphosphate (IP,)] in 0.1 M formic acid, essentially as described (Bemdge, 1983; Batty et al., 1985). After each step, the columns were washed with an additional 10 ml of the corresponding solution. Using this procedure, recovery of the inositol phosphates was >95% for IP, >87% for lPz. and >75% for IP3. Radioactivity in each sample (2.5 ml) eluted from the columns was quantitated by mixing with 10 ml of ACS scintillation fluid and measuring the radioactivity with a Beckman LS-230 liquid scintillation counter.

Measurement of DG For the determination of DG, stock PC12 cells were seeded and grown in Belco T150 flasks. When the cells reached 5070% confluence, the growth medium was removed and the cells were preincubated for 15 rnin in PBS (or for 4 h in HEPES-buffered DME) before the addition of growth factors. NGF and bFGF (dissolved in incubation medium) were added directly to the flasks, and at the indicated time the medium was removed and 3 ml of ice-cold methanol was added immediately. The cells were then scraped into the methanol using a Teflon scraper, and the flasks were washed with an additional 3 ml of methanol, which was collected into glass tubes into which 3 ml of chloroform was added to extract cellular phospholipids. The tubes were vortexed frequently, and extraction was carried out for 45 rnin at room temperature. The aqueous and chloroform phases were separated essentially as described (Bligh and Dyer, 1959; Preiss et al., 1986) by adding another 3 rnl of chloroform and 3.5 ml of 1 MNaCl, and then centrifuging briefly. The chloroform layer (containing the total lipid-soluble extract) was removed into another glass tube. The precipitate was extracted for an additional 45 rnin and, after separating the phases, the chloroform extracts were combined. With this procedure, the recovery of DG, as determined using 1,2-diolein, was always in excess of 95%. A small aliquot of this extract was removed for determination of total lipid phosphorus (Ames and Dubin, 1960), and the remainder was dried under a stream of N2. The samples were stored (usually for 1-3 days) under N2 at -7O"C, and then were dissolved in HPLC solvent (see below) before determination of the DG content by HPLC. The amount of DG in each sample was measured by applying it to an HPLC system with a Resolve Silica column (Waters Associates) using an isocratic solvent system consisting of hexane/isopropanol/acetic acid (100:0.5:1) at a flow rate of 2 ml/min. This method gives an accurate determination of DG which reproducibly elutes as a bimodal peak 1 1 rnin after injection ofthe sample as detected by measuring the absorbance at a wavelength in the range 190-212 nm (Hamilton and Comai, 1984; Bocckino et al., 1985; Abe and Kogure, 1986). Measurements were made with an HP-1090 HPLC system (Hewlett Packard) coupled to a variable wavelength UV detector (Hitachi) model L-4200; maximum sensitivity for DG was obtained at 196 nm. The identity of the DG peak was confirmed by TLC, and quantitation was achieved using 1,2-diolein as standard. The amount of 1,2diolein was proportional to the area of the peak in the range 1-50 fig. The total amount of DG present in the cellular extracts was expressed as a fraction of total lipid phosphorus (mol %).

J. Neurochem.. Vol. 54. No. 5, 1990

RESULTS Comparison of the effects of NGF, bFGF, and CarbdChOl on the production of inositol phosphates in PC12 cells Prcliminary experiments indicated the production of inositol phosphates by NGF was significant and reproducible only when the hormone was added directly to [3H]inositol-labeledPC12 cells that were left attached to the substratum (Belco plastic tissue culture flasks, with or without collagen coating). The response obtained in cells that were removed from the substrate was small and not always reproducible (not shown), suggesting either that the removal of the cells from the substratum disrupts them in a way that reduces their responsiveness to NGF, or, alternatively, that the mechanism involved in the generation of inositol phosphates by NGF is itself dependent on cell attachment. This is consistent with a previous study showing that NGF-induced stimulation in the incorporation of [32P]04into phosphatidylinositol is greater in attached PC12 cells than in cells in suspension (Traynor et al., 1982). It is also noteworthy that NGF-induced neurite outgrowth requires cell attachment (Schubert and Whitlock, 1977; Fujii et al., 1982) and is inhibited by antibodies to a cell surface fibronectin receptor (Schwartz et al., 1988). Subsequent studies therefore were always conducted on cells that were grown on, and lcft attached to, a suitable substrate. As shown in Figs. 1 and 2, the addition of NGF to [3H]inositol-labeledPC12 cells that were grown on, and left attached to, Belco flasks and preincubated for 15 rnin in the presence of 10 mM LiC1, induced maximal increases of 118, 140, and 50% in IP, IP2, and IP3, respectively, within 30 min. 'There was no detectable change in the level of IP4 (results not shown). Similar increases were obtained when the experiments were conducted using flasks that had been collagen-coated (not shown). These increases are comparable to those previously reported (Contreras and Guroff, 1987).It is noteworthy, however, that although the levels of IP2 and IP3 both showed significant percentage increases relative to the control, the bulk of the label (approximately 85%) was recovered in IP; about 10% was recovered in IP2 and 5% in IP3 (Fig. 2). bFGF stimulated measureable changes in the production of inositol phosphates only when added at doses of around 50 ng/ ml (Fig. lb). At these concentrations [bFGF (50 ng/ ml) and NGF (200 ng/ml)], the two hormones were similar in their ability to stimulate the production of inositol phosphates (see Fig. 2). In the absence of 10 m M LiCl, significant increases in the production of inositol phosphates induced by NGF and bFGF could be detected at early times (1 -5 rnin), but no significant change could be detected after 30 rnin of continuous stimulation (data not shown). The muscarinic agonist carbachol is purported to elicit its effects by stimulating phosphoinositide hydrolysis in PC12 cells (Vicentini et al., 1986). In this sys-

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1.2-DIACYLGLYCEROL PRODUCTION BY NGF A N D bFGF

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level in PC 12 cells growing in DME supplemented with 10% fetal calf serum and 5% heat-inactivated horse serum was 6.2 ? 1.3 mol %. The level of DG in PC 12 cells after the growth medium was removed and replaced with DME (without serum supplementation) was 2.52 k 0.75 mol % after a 15-min incubation, and thereafter remained at about this level for at least 6 h. Because PC 12 cells are known to depend on the presence of serum and/or certain growth factors for survival (Greene and Tischler, 1982), we did not measure DG levels after longer times under serum-free conditions. These results suggest that the “resting level” of DG in PC I2 cells when incubated in DME (in the absence of serum) may be close to an order of magnitude higher

. 15

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Time after addition (min) FIG. 1. The stimulation of 3H-inositol phosphates by NGF and bFGF. PC12 cells were prelabeled with [3H]inositolfor 40-48 h in DME supplemented with 5% fetal calf serum and 2.5% heat-inactivated horse serum. After three washes with PBS, the [3H]inositol-labeledcells were incubated in PBS containing 0.9 mM Ca2’ and 10 mM glucose in the presence 10 mM LiCI. After 15 min, NGF at 200 ng/ml (a)and bFGF at 50 ng/ml (b) were added, and at various times after the addition of the growth factors, the [3H]IP2(O),and [3H]IP3(m) was determined formation of f‘H]IP (e), as detailed in Experimental Procedures. The data are expressed as a percentage change from the control value (defined as the level of inositol phosphates at time zero in the absence of any growth factor). Only the time course for [3H]IP in the control is shown (0).Each point represents the mean of between three and five independent experiments; SEMs were omitted for clarity, but never exceeded 8% of the data.

tem, the production of inositol phosphates by 0.5 mM carbachol was 5- 10-fold greater than that induced by NGF and bFGF under otherwise identical experimental conditions (see Fig. 2). Importantly, carbachol at this concentration has been reported not to induce neurite outgrowth in PC12 cells (Schubert et al., 1978); in our hands, it appeared to stimulate proliferation of PC 12 cells after just 1-2 days of continuous treatment (data not shown). Total mass levels of DG in PC12 cells As shown in Table 1, the mass level of DG of PC12 cells attached to Belco plastic flasks depended markedly on the medium in which the cells were incubated. The

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Time after addition (min) FIG. 2. Time course of the stimulation in production of inositol

phosphates by NGF. bFGF, and carbachol. PC12 cells were prelabeled with [3H]inositol and incubated in PBS as described in the legend to Fig. 1 . The curves in a, b, and c show the increase in [3H]IP, [3H]IP2,and [3H]IP3,respectively, at various times after the addition of 200 ng/ml NGF (O), 50 ng/ml bFGF (m), 0.5 mM carbachol (@), or an equivalent volume of PBS for the control (0). The data are presented in cpm. Each point represents the mean of between three and five independent experiments; SEMs were omitted for clarity, but never exceeded 10% of the data.

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J. G. ALTIN AND R. A . BRADSHAW TABLE 1. Total DG ofPC12 cells incubated in different media Incubation

Total DG, mol %

DME 10%FCS + 5% HS DME After 15 rnin After 4 h DME HEPES-buffered After 15 rnin After 4 h DME + 3 mM EGTA for 15 rnin PBS After I5 rnin After I h PBS HEPES After 15 rnin After 1 h

6.21 i 1.30 ( 5 )

+

+

2.52 f 0.75 (4) 2.12 k 0.52 ( 5 ) 3.42 f 0.64 (4) 2.78 k 0.49 (4) 0.75 f 0.10 (3) 0.62 f 0.08 (6) 1.10 f 0.13 ( 5 )

The lowest level of total DG in PC12 cells (0.62 mol %) was obtained after a 15-min preincubation of the

cells in PBS plus 10 mM glucose (Table 1). This condition (i.e., incubation in PBS plus glucose, hereafter referred to as incubation in PBS) was considered preferable for measuring the effect of NGF and bFGF on DG production. It should be pointed out that there was a considerable increase in the level of total DG when the PC 12 cells were incubated in PBS for periods longer than about 45 min. In fact, DG levels increased 70430% after about 1 h of incubation (Table 1). This increase was attributed to an acidification of the medium, because in these experiments the pH of the me-

0.98 k 0.15 (4) 1.24 k 0.21 (4)

PC I2 cells were grown in DME supplemented with 10%fetal calf serum (FCS) and 5% heat-inactivated horse serum (HS) as detailed in Experimental Procedures. For each set of incubation conditions, the cells were fed fresh growth medium 24 h beforc the experiment. For the first condition, in which the experiment was conducted in growth medium, the medium was simply removed and methanol was added immediately to fix the cells. Similarly, for all other incubation conditions, the growth medium was removed, and the cells were washed once with the indicated medium before incubating in this medium for the appropriate time and adding methanol. In each instance, the mass level of DG was determined by HPLC as outlined in Experimental Procedures. Each value represents the mean f SEM of the number of independent measurements shown in parentheses.

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than that reported for serum-starved IIC9 fibroblasts (Raben et al., 1987; Wright et al., 1988) and human skin fibroblasts (van Veldhoven and Bell, 1988), but similar to that reported for serum-starved A431 cells (van Veldhoven and Bell, 1988). The mass level of DG of PC12 cells was found to be affected by alterations in the pH of the extracellular medium (see below). In the experiments camed out in bicarbonate-buffered DME, the cells were equilibrated in an atmosphere of 95% air/5% C 0 2 until the medium was removed and methanol was added to prepare the cells for the chloroform/methanol extraction (see Experimental Procedures). Under these conditions, the pH of the medium removed from the cells was always in the range 7.3-7.5 as measured before significant evaporation of COz had occurred. The use of HEPESbuffered DME (containing 20 M H E P E S ) , or the addition of 20 mM HEPES to the PBS, always gave a slightly higher “basal” level of DG in PC 12 cells than the use of media in which the HEPES was omitted (see Table 1). It is interesting that the addition of 3 m M EGTA to reduce the Ca2+concentration of the DME to submicromolar levels resulted in lowering of DG to a level comparable to that observed after the cells were preincubated for 15 rnin in PBS. As high levels of DG are usually associated with cellular activation, it would appear from these data that when incubated in DME, PC12 cells are “activated” or stimulated to produce endogenous DG even in the absence of added serum or growth factors. J. Neurochem., Vol. 54, No. 5, 1990

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FIG. 3. Stimulation of DG production by NGF, bFGF, and carbachol. PC12 cells were grown in DME supplemented with 10% fetal calf serum and 5% heat-inactivated horse serum. In a, the medium was removed, and the cells were washed once with PBS containing 10 mM glucose and then preincubated in this medium for 15 rnin before the addition of 200 ng/ml NGF (O), 50 ng/ml bFGF (m), or 0.5 mM carbachol(0).In the control experiments(0),an appropriate volume of PBS was added. After incubating in the presence of these agents for the indicated times, methanol was added and DG was extracted and assayed as described in Experimental Procedures. The data in b were obtained from similar experiments with NGF, bFGF, or control with the exception that the cells were preincubated in DME (without added serum) instead of PBS. Each point represents the mean of between three and five independent determinations. Only the SEMs (bars) for NGF and control data are shown for clarity; these errors were typical of those obtained in the data for bFGF and carbachol.

1,2-DIACYLGLYCEROL PRODUCTION BY NGF AND bFGF

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FIG. 4. Dose response for the productionof DG by NGF and bFGF. The experimental conditions for these measurements were similar to those described in the legend to Fig. 3a. PC12 cells previously grown in DME supplemented with 10% fetal calf serum and 5% heat-inactivated horse serum were preincubatedfor 15 min in PBS containing 10 mM glucose. After the preincubation period, either or bFGF (@) was added at the indicated concentrations. NGF (0) Determination of the mass level of DG was carried out 15 rnin after incubation with the indicated concentrations of growth factor. Other details are outlined in Experimental Procedures. Each point represents the mean f SEM (bars) of between four and six independent experiments.

dium had fallen from 7.4 initially, to about 6.7 after a 1-h incubation (data not shown). Moreover, a similar increase in DG occurred even after a shorter (1 5-30 min) incubation when the pH of the PBS was adjusted to 6.8 before the incubation (see below). This suggests that the level of DG in PC12 cells is increased significantly after a decrease in the pH of the extracellular medium, and that prolonged incubation (>45 min) in PBS media should be avoided to reduce the possibility of artifactual production of DG by PC12 cells when studying the effects of growth factors. The addition of NGF, bFGF, or carbachol for periods of 45 rnin or less did not induce any further change in the pH of the extracellular medium (not shown). Stimulation of DG production by NGF, bFGF, and carbachol The mass level of DG in PC12 cells incubated in PBS was increased significantly by the addition of 200 ng/ml NGF and also by 50 ng/ml bFGF (Fig. 3a). At these concentrations, the increase in DG induced by the two growth factors was similar and occurred in two phases. First, there was a rapid twofold increase in the level of DG, which peaked at 1-2 min. This was often followed by a slight decline, and then a slower, more sustained increase to two- to threefold (at 15 min). This level decreased slightly after 30 rnin of growth factor addition, suggesting that as well as promoting DG release, these growth factors stimulate DG metabolism, presumably through the action of a DG lipase or by

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conversion to phosphatidic acid (Berridge, 1984). As can be seen from the dose-response curves in Fig. 4, half-maximal stimulation of DG production by NGF and bFGF occurs at concentrations of about 25 and 4 ng/ml, respectively. These concentrations are well within the range known to stimulate neurite outgrowth in PC12 cells (Rydel and Greene, 1987). As the production of inositol phosphates induced by NGF and bFGF was considerably smaller than that induced by carbachol (see Fig. 2), the effect of carbachol on the production of DG also was measured. The data in Fig. 3a show that under conditions where the production of inositol phosphates induced by carbachol was 5- 10-fold greater than that induced by NGF and bFGF, the production of Ix;by carbachol was similar to that induced by NGF and bFGF. Because neurite outgrowth in unprimed PC12 cells exposed to either NGF or bFGF usually takes 1-2 days, studies with these growth factors are usually conducted with cells incubated in DME rather than in a basic salt solution like PBS. It was important, therefore, to determine whether the increase in DG induced by NGF and bFGF in PC12 cells incubated in PBS occurred also when the cells were incubated in DME. Although the “basal” level of DG was higher in cells incubated in DME (see Table l), and in some experiments appeared to cause some ‘‘saturation’’ of the response that could be induced by the growth factors, in most experiments the addition of NGF and bFGF to cells incubated in DME (without serum) resulted in essentially identical responses (see Fig. 3b). Clearly, both NGF and bFGF stimulate the production of DG in PC12 cells under conditions in which neurite outgrowth is known to occur. Dependence of DG production on extracellular Ca2+ and K+ and the acidification of the extracellular medium The data in Fig. 5a show that a 5-min preaddition of 1.5 mM EGTA to PC12 cells incubated in PBS completely abolished the effects of NGF and bFGF on the production of DG. A similar inhibition was observed when the cells were preincubated for 15 rnin in PBS containing no added Ca2+, but instead, 50 pM EGTA (not shown), indicating that the production of DG by NGF and bFGF is dependent on the presence of extracellular Ca”. In addition, as shown in Fig. 5b, a Ca2+-dependentincrease in the mass level of DG in PC12 cells (about 80% increase within 30 min) could be induced by exposing the: cells to a depolarizing concentration (50 mM) of K+ (by substitution of an equivalent amount of NaCY), which is known to stimulate Ca2+influx through voltage-sensitiveCa’+ channels. The incubation of PC: 12 cells for 30 rnin in PBS at pH 6.8 (adjusted with HC1 before the incubation) resulted in about a doubling of the “basal” DG level in comparison with PC12 cells incubated in PBS at pH 7.4 (Fig. 5b). The reason for this is unclear, but it presumably reflects the sensitivity of the various phosJ. Neurochem., Vol. 54, No. 5. 1990

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J. G. ALTIN AND R. A . BRADSHAW 2.0

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K+

pH 6.8

FIG. 5. Effect of lowering the extracellularfree Ca2+concentration on DG productionby NGF and bFGF, and the effect of acidification of the extracellular medium and exposure to high extracellular K+. In a, PC12 cells were grown in serum-supplemented DME as described in Experimental Procedures and then incubated in PBS containing 10 mM glucose for 15 min before the addition of either NGF (200 ng/ml) or bFGF (50 nglml), or an equivalent volume of PBS (control, filled columns) as indicated. In some experiments (hatched columns), 1.5 mM EGTA was added 5 min before the addition of the growth factors. In each instance, determinationof the mass level of DG was carried out after 15 min of incubation with the respectivegrowth factor. In b, the cells were preincubated in PBS for 15 min, and then this medium was removed and the cells incubated in either PBS modified by substitution of 50 mM KCI for an equivalent amount of NaCI, or PBS adjusted to pH 6.8 with HCI, as indicated. Similarly, hatched columns represent experiments in which 1.5 mM EGTA was included throughout the incubation. In these experiments, DG was measured after 30 rnin of incubation with the respective medium. Each value represents the mean k SEM of three or four experiments conducted in duplicate. "p < 0.05 and ' p < 0.001, compared with the respective value in the absence of EGTA, by Student's t test.

pholipases involved in production of DG to pH, or to some pH-dependent event such as Ca2' transport across the plasma membrane (Altin and Bygrave, 1988). Effect of P A 0 and MTA on DG production The effect of inhibitors that have been reported to interfere with neurite outgrowth and/or some aspect of receptor function on NGF- and bFGF-stimulated production of DG in PC12 cells also was examined. The data in Fig. 6 show that whereas PA0 (25 p M ) does not significantly alter the basal level of DG in cells incubated in PBS, it completely inhibits DG production stimulated by NGF and bFGF. The data in Fig. 6 also show that the methyltransferase inhibitor MTA significantly inhibited (approximately 90%inhibition) DG production induced by NGF, but only slightly inhibited (20-30% inhibition) DG production induced by bFGF.

DISCUSSION NGF and bFGF, though different in potency, induce similar increases in inositol phosphates in PC 12 cells. J. Neurochem., Vol. 54, No. 5, 1990

These increases, however, are close to an order of magnitude smaller than those induced by carbachol (under otherwise identical conditions), an agonist that stimulates the hydrolysis of phosphoinositides and the mobilization of cellular Ca2+(Pozzan et al., 1986; Vicentini et al., 1986). Our observations that the action of NGF is accompanied by relatively small changes in inositol phosphates, and that the major product is not IP3, but IP (which is ineffective in mobilizing intracellular Ca2+), are consistent with reports by other workers that Ca2' flux changes induced by NGF in PC12 cells are small (Pandiella-Alonso et al., 1986) and often escape detection (Landreth et al., 1980; van Calker et al., 1989). The relatively small effect of NGF and/or possible differences in cell lineage and experimental procedures may well explain these findings. In some instances, NGF has been reported to induce a phosphoinositide response when added in conjunction with other agonists, or to potentiate the effects of growth factors like bradykinin and epidermal growth factor (Volante et al., 1988; van Calker et al., 1989). Whether the relatively small stimulation of phosphoinositide hydrolysis induced by NGF or bFGF in PC 12 cells is necessary, or plays a direct role in mediating neurite outgrowth, is unknown. Indeed, Ca2' levels have been shown to be raised in the growth cones of extending neurites in Helisoma neurons (Cohan et 2.0

C C

1.5

1.o

0.5

0.0

control

NGF

bFGF

FIG. 6. Inhibitionof DG production by PA0 and MTA. PC12 cells previously grown in DME supplemented with 10% fetal calf serum and 5% heat-inactivatedhorse serum were preincubated for either 15 or 30 min in PBS in the presence of MTA (3 mM, shaded columns) or PA0 (25 y M , hatched columns), respectively. Subsequently, in some experiments 200 ng/ml NGF or 50 ng/ml bFGF was added as indicated. Similar experiments also were conducted in the absence of inhibitors (filled columns). The mass level of DG was determined after a 15-min incubation in either the presence or absence of growth factor as indicated. Each value represents the mean t SEM of three determinations performed in duplicate. 'p i0.10, "p < 0.02, and "p < 0.001, each compared with the respective control value by Student's t test.

1,2-DIACYLGLYCEROL PRODUCTION BY NGF AND bFGF al., 1987), and the phosphoinositol cycle has been implicated in the assembly of actin filaments and their anchorage to cellular membranes in platelets (Burn, 1988). In addition, Ca2+appears to be required for the attachment of PC12 cells to substrate (Schubert et al., 1978), an event that is necessary for the induction of neurite regeneration by NGF (Greene and Tischler, 1982). It is possible, therefore, that phosphoinositide hydrolysis (e.g., changes in Ca2+flux) may be important in the elaboration of neurites once the cells have been “primed”. However, because carbachol (an agent that induces a considerable stimulation of phosphoinositide hydrolysis) does not itself induce “priming” or neurite outgrowth in PC12 cells (Schubert et al., 1978), the stimulation of phosphoinositide hydrolysis (and the associated production of inositol phosphates and DG) is not itself a sufficient condition for the induction of either response. It would seem that some other signal(s) generated from the binding of NGF or bFGF to their specific receptors must also be required in mediating the neurotrophic response, or, alternatively, that the large increase in inositol phosphates (with respect to DG production) induced by carbachol is inhibitory to neurite outgrowth. A major finding from this study is that NGF and bFGF induce the production of DG when added to PC12 cells in culture. This is the first direct demonstration of an increase in DG following stimulation of PC12 cells with these growth factors. Because DG is an activator of protein kinase C, these findings are consistent with the observations of others that protein kinase C activity is increased following the stimulation of PC12 cells with NGF (Cremins et al., 1986; Hama et al., 1986, 1987). Furthermore, the data strongly suggest that protein kinase C also is activated following the stimulation of PC 12 cells with bFGF. Our data show that DG production stimulated by NGF and bFGF was comparable to that stimulated by carbachol (Fig. 3a); in contrast, the production of inositol phosphates induced by carbachol was 5- I0 times higher (see Fig. 2). As measurement of inositol phosphates uses isotopic labeling, the stoichiometry of inositol phosphates to DG production cannot be determined directly from these data. Although there is some evidence that different agonists may differentially regulate the metabolism of inositol phosphates and/or the net synthesis or metabolism of DG in some systems, no difference in the effects of NGF, bFGF, or carbachol on the metabolism of these messengers in PC12 cells has been reported. One interpretation of these results is that NGF and bFGF each stimulate the production of DG primarily from the hydrolysis of phospholipids other than the phosphoinositides. This conclusion is in agreement with a recent study using [32P]NMR which showed that NGF induces rapid changes in the levels of phosphatidylcholine and phosphatidylethanolamine in PC12 cells (Miccheli et al., 1989) and is consistent with similar observations in other cells (Bocckino et al., 1985; Saltiel et al., 1987; Polverino

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and Barritt, 1988; Rosoff et al., 1988; Saltiel and Cuatrecasas, 1988). In this regard, it is noteworthy that the actions of interleukin- 1 in T-lymphocytes (Rosoff et al., 1988), and of insulin in BC3Hl cells (Saltiel et al., 1987), are similar to those of NGF and bFGF in PC 12 cells in that the effects are not accompanied by large increases in either inositol trisphosphates or changes in Ca2+flux. A very recent study, in fact, shows that in PC 12 cells, NGF (analogous to the action of insulin in BC3H1 cells) stimulates the hydrolysis of glycosylphosphatidylinositol, leading to the production of a species of DG that is labeled with [’Hlmyristate and an inositol phosphate glycan (Chan et al., 1989). Although it is possible that the increase in myristate-labeled DG observed by Chan et al. (1989) at 1-2 min of stimulation contributes significantly to the initial increase in mass level of DG observed in this work, the isotopic labeling technique employed by Chan et al. (1989) to measure DG does not allow us to assess how much of the DG produced may be accounted for by this mechanism. We are presently determining the nature of the fatty acid moieties on the DG produced by the action of NGF, bFGF, and carbachol in PC 12 cells to identify the phospholipid progenitors involved. Because the production of inositol phosphates by NGF (Contreras and Guroff, 1987) and the production of DG demonstrated here is Ca2+-dependent, but the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C is considered not to be (Renard et al., 1987), the possibility exists that the phospholipase(s) activated by NGF or bFGF is specific for phosphatidylinositol, or, perhaps, for some other species of phospholipids, rather than for phosphatidylinositol4,5bisphosphate. Inositol phosphate production also may occur either directly or indirectly through the activation of a phospholipase D (Bocckino et al., 1987; Balsinde et al., 1988). It remains to be established, however, whether the primary effect of the occupation of the NGF or bFGF receptors in PC 12 cells is the activation of a phospholipase(s) [through a mechanism involving a GTP-binding protein (Gilman, 1987; Casey and Gilman, 1988; Fain et al., 1988)], or whether activation of the phospholipase(s) is secondary to the stimulation of Ca2+ influx through some Ca2+ channel. Thus, it may be possible that, as has been reported for isolated hepatocytes (Bocckino et al., 1985; Polverino and Barritt, I988), phosphoinositide hydrolysis serves as a trigger for the stimulation of Ca2+influx to activate other phospholipases for the generation of DG and the activation of protein kinase C , and also, perhaps, for the generation of other important second messenger signals. Alternatively, there may be a more direct effect of the receptor mechanism (perhaps involving a GTPbinding protein) on the activation of a specific phospholipase C different from the phosphoinositide-specific phospholipase C (Saltiel et al., 1987; Rosoff et al., 1988; Saltiel and Cuatrecasas, 1988). As depolarization of the PC 12 cell plasma membrane by exposure to high extracellular K+ stimulates DG production (Fig. 5 ) and J. Neurochem.. Vol. 54, No. 5. 1990

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phosphoinositol turnover (Traynor, 1984), presumably through the activation of voltage-sensitive Ca2+channels, and as phosphoinositide hydrolysis, including perhaps the associated production of phosphatidic acid, has been proposed to be involved in the stimulation of Ca2+influx in other systems (reviewed in Altin and Bygrave, 1988), it is possible that in PC12 cells either mechanism is involved. Our finding that DG production can be inhibited by P A 0 is consistent with the evidence that physiological responses are mediated by type-1 (slow) NGF receptors (Sonnenfeld and Ishii, 1982; Stach and Wagner, 1982; Eveleth and Bradshaw, 1988) and that in PC12 cells the stimulation of DG production by NGF occurs primarily through an interaction with this receptor type. However, the ability o f P A 0 to inhibit also DG production induced by bFGF (and also by carbachol, data not shown) suggests that its action may be at a common point of the signal transduction pathway (e.g., a GTPbinding protein, or a Ca2+ channel) coupling the receptors to activation o f phospholipase C, or perhaps phospholipase C itself. In the present system, the effect of this agent is not specific enough to be explained solely in terms of an inhibition of receptor internalization (Knutson et al., 1983) or an inhibition of the interconversion of the different receptor types (Eveleth and Bradshaw, 1988). The inhibition of the NGF-induced D G production by 3 m M MTA, the methyltransferase inhibitor, is consistent with the reported effects of MTA on a number of other responses mediated by NGF. In PC 12 cells, MTA has been reported to inhibit NGF-induced stimulation o f neurite outgrowth, ornithine decarboxylase activity, and protein phosphorylation (Seeley et al., 1984). Moreover, MTA has been reported to inhibit the rapid redistribution of F-actin induced by NGF (Paves et al., 1988). Our finding that MTA is less effective in inhibiting the increase in DG induced by bFGF may indicate a fundamental difference in the mechanism of these two hormones in the cell. It is not certain that activation of protein kinase C plays a role in stimulation of neurite outgrowth by NGF and bFGF in PC 12 cells. Although a number of studies support such a role, a recent study reports that NGF still elicits neurite outgrowth in PC12 cells in which protein kinase C has been down-regulated by prolonged treatment with phorbol esters (Reinhold and Neet, 1989). Our present observation that carbachol, an agent that does not induce neurite outgrowth, also stimulates the production of DG suggests either that the activation of protein kinase C is not sufficient for stimulating neurite outgrowth, or that different forms of protein kinase C may be involved. It is possible, for example, that different forms of the enzyme are regulated differentially by DGs of different fatty acid composition. Acknowledgment: This work was supported by USPHS research grant NS 19964, program project grant AG00538, a n d American Cancer Society research grant BC273.

J . Nwruchrm., Vol. 54, N u 5. 1990

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