Journal of Neuroscience Research 2571-80 (1990)
Phorbol Ester-Induced Change in Astrocyte Morphology: Correlation With Protein Kinase C Activation and Protein Phosphorylation B.C. Harrison and P.L. Mobley Department of Pharmacology, University of Texas Health Science Center, San Antonio
Treatment with 300 nM phorbol 12-myristate 13-acetate (PMA) transforms polygonal-shaped cultured astrocytes into process-bearing cells and produces a shift in protein kinase C (PK-C) from the cytosol to the membrane. Exposure to PMA also produces increases in the phosphorylation of several proteins including vimentin, glial fibrillary acidic protein (GFAP), an acidic 80,000 molecular weight protein, and two 30,000 molecular weight proteins (PI5.5 and 5.7). The effects of PMA on the translocation of PK-C and on protein phosphorylation precede the PMAinduced changes in astrocyte morphology, and a close correlation exists between the concentration of PMA necessary to elicit half-maximal and maximal effects on the shift of PK-C to the membrane and on protein phosphorylation. In addition, the PMA-induced alterations in cell morphology are not permanent, and within 24 hr after PMA treatment the cells have reverted almost to their original morphology. A second exposure to PMA at this time fails to elicit further change in cell shape and is also incapable of producing increases in the phosphorylation of proteins. It was determined that there is little, if any, PK-C present in these PMA-pretreated cells. The morphological responsiveness to PMA gradually returns in 5 to 8 days after the initial treatment with PMA, and this is accompanied by the recovery of PK-C activity and the phosphorylation response. Therefore, these studies suggest that the effect of PMA on astrocyte morphology is mediated by the activation of PK-C and subsequent protein phosphorylation. Key words: glia, GFAP, vimentin
be conducted across the cell membrane to regulate cell function. One such system involves the generation of inositol trisphosphate and diacylglycerol from the hydrolysis of phosphatidylinositol (Friedel et al., 1969; Berridge, 1984; Nishizuka, 1984). lnositol trisphosphate promotes the release of calcium from intracellular stores (Streb et al., 1983; Berridge and Irvine, 1984) while diacylglycerol is capable of activating the calcium and phospholipid-dependent protein kinase, protein kinase C (PK-C) (Takai et al., 1979; Kishimoto et al., 1980). PK-C appears to have a ubiquitous distribution in animal tissue and has been reported to be present in cultures of rat astrocytes (Mobley et al., 1986; Neary et al., 1986). It has also been demonstrated that the PK-C activator, phorbol 12-myristate 13-acetate (PMA), produces changes in astrocyte morphology such that flat, polygonal-shaped cells are transformed into stellate, process-bearing cells (Mobley et al., 1986). PMA has also been reported to change the morphology of other cell types including fibroblasts (Diamond et al., 1974), the epithelial cell line MDCK (Ojakian, 1981), and macrophages (Phaire-Washington et al., 1980). Besides eliciting alterations in cell shape, treatment of astrocyte cultures with PMA was found to produce a rapid translocation of PK-C activity from the cytosol to the membrane of these cells (Mobley et al., 1986; Neary et al., 1988). Previous studies have also demonstrated that treatment of astrocytes with 300 nM PMA produces increases in the 32P incorporation into several phosphoproteins including an 80,000 (PI 4.5) and two 30,000 (PI 5.5 and 5.7) molecular weight proteins and the cytoskeletal proteins glial fibrillary acidic protein (GFAP) and vimentin (Mobley and Harrison, 1987; Harrison and Mobley, 1989). While treatment of astrocytes with PMA induces
INTRODUCTION Extracellular signals such as neurotransmitters and hormones can induce the generation of signal messengers that activate protein kinases. In turn, protein kinases are capable of regulating the phosphorylation of proteins, thereby establishing a means by which information can 0 1990 Wiley-Liss, Inc.
Received July 12, 1989; revised September 14, 1989; accepted September 15, 1989. Address reprint requests to Philip L. Mobley, Ph.D., Department of Pharmacology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284.
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changes in astrocyte morphology and protein phosphorylation, it is not certain if these two effects are related to each other or to what extent these effects are mediated by PK-C. In the present studies we have attempted to correlate the effects of PMA on astrocyte morphology with changes in PK-C activation and subsequent protein phosphorylation. Also, the effects of PMA on morphology and protein phosphorylation have been studied in PKC-depleted astrocytes. The results indicate that the effects of PMA on astrocyte morphology correlated with a shift of PK-C to the membrane fraction and an increase in protein phosphorylation of several proteins. Additionally, in PK-C-depleted cells PMA lost its ability to induce changes in cell morphology or protein phosphorylation. A substantial amount of PK-C activity returned within 5 to 8 days after the initial exposure to PMA, and this correlates with a return of the morphological and phosphorylation response to this agent.
EGTA, 2 mM EDTA, 1 mM dithiothreitol, 0.1 mgiml leupeptin, 0. I mM phenylmethylsulfonyl fluoride, pH 7.5) and placed on ice. Cell suspensions were then centrifuged for 1 min at 120,000g in a Beckman Airfuge. The supernatant (soluble extract) was saved and the resulting pellet was resuspended in Tris buffer and centrifuged. The supernatant was discarded and the pellet was resuspended in Tris buffer 0.1% Triton X-100 and placed on ice for 30 min. The suspension was centrifuged and the resulting supernatant (membrane extract) saved. The level of PK-C activity was determined in both the soluble and membrane fractions as described previously (Kikkawa et al., 1982) by using histone (Type III-S; Sigma Chemical Co., St. Louis, MO) as a substrate. For experiments where the PMA-induced loss and subsequent recovery of total PK-C activity was determined, cells were homogenized directly in buffer containing 0.1% Triton X- 100 and the total homogenate was assayed for PK-C. The total assay volume was 50 pl and contained (final concentration) 20 mM Tris-HC1 (pH MATERIALS AND METHODS 7 . 3 , 2 mM EGTA, 0.4 mM EDTA, 0.2 mM dithiothreCell Culture itol, 20 pg/ml leupeptin, 0.02 mM phenylmethylsulfonyl P , pg histone, and 10 (1.1 Astrocyte cultures were prepared from the neocor- fluoride, 100 pM [ Y - ~ ~ P I - A T40 tex of 1 day-old Sprague-Dawley rats. The tissue was tissue preparation. The reaction was conducted at 30°C sliced at 400 pm intervals with a McIlwain Tissue Chop- for 3 min in the presence or absence of 1.5 mM CaCI,, per and placed in sterile Krebs Ringer bicarbonate buffer 3 mM Mg-acetate, 20 p g phosphatidylserine, and 0.3 pg (pH 7.4) with trypsin (2500 unitsiml) for 5 min at 37°C. diolein (98% 1,2 isomer; Sigma Chemical Co., St. Medium containing fetal bovine serum was then added to Louis, MO). Aliquots of the reaction mixture were inhibit the trypsin. The cells were pelleted and then trit- placed on filter paper squares ( 1 .S cm; Whatman 3MM) urated in calcium and magnesium-free Krebs Ringer bi- and immersed in 10% trichloroacetic acid (TCA) to tercarbonate buffer containing 0.0 1 mgiml DNase (deoxy- minate the reaction. After washing the filter paper ribonuclease 1 ; Boehringer Mannheim, Indianapolis, squares in boiling 5% TCA (twice), ethanol, and acetone IN). The dispersed cells were transferred to Dulbecco’s (twice), radioactivity was determined by Cerenkov modified Eagle’s medium with high glucose (DMEM; counting in water. pH 7.4) containing 10% fetal bovine serum and then filtered through a 70 pm nylon screen and plated (300,000 cells/cm2) in tissue culture flasks (25 cm2). The Protein Phosphorylation Confluent cultures were washed with sterile 0.9% astrocytes were grown to confluence in DMEM plus serum in an atmosphere of 95% air/S% CO, at 37°C. NaCl followed by phosphate-free DMEM, and then 1 ml Contaminating cells were removed by orbital shaking of phosphate-free DMEM containing 5% dialyzed calf (McCarthy and de Vellis, 1980) and the astrocytes were serum was added to each dish. An aliquot of ’2P-orthosubcultured into 35 mm dishes (30,000 cells/cm2) and phosphate (0.1-0.2 mCi; approximately 285 Ciimg) was maintained as described above. Based on morphology added to each dish and after a 2 hr labeling period, the and immunocytochemical studies (peroxidase-antiperox- cultures were exposed to either 300 nM PMA or the idase staining for GFAP) these cultures were relatively dimethyl sulfoxide vehicle (DMSO, 0.05%) for 1.5 min. free of contaminating cells (greater than 9.5% astrocytes). After treatment, the culture medium was removed and Cultures were then used for experiments within 14 days the cells were washed once with ice-cold PBS and then dissolved in 1% sodium dodecyl sulfate (SDS). An aliafter reaching confluence. quot of this solution was then added to 9 volumes of Protein Kinase C Assay ice-cold acetone. The precipitate was collected by cenThe culture medium was removed and the cells trifugation and dissolved in urea-containing lysis buffer were washed twice with ice-cold phosphate-buffered sa- (O’Farrell, 1975). Equal volumes (30 pl; approximately line (PBS). The cells were scraped from the dishes and 60 pg protein) of the samples were then subjected to homogenized in Tris buffer (20 mM Tris-HCI, 10 mM 2-dimension gel electrophoresis (O’Farrell, 1975). The
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Phorbol Esters and Gstrocyte Morphology
protein content of the culture dishes and 32P incorporation are quite uniform such that the samples contained approximately equal amounts of protein and acidiorganic solvent insoluble radioactivity. After electrophoresis, the gels were fixed in a solution of 10% acetic acid and 10% isopropanol and dried, and autoradiograms were prepared by using x-ray film (Kodak XAR-5) with or without Dupont Lightning Plus intensifying screens as necessary. Computer images of the protein spots on the autoradiograms were made by using a Grinnell imageprocessing system coupled to a VAX-111750 computer. The degree of exposure of each spot of interest was determined based on the gray level of each pixel that comprised the spot image.
Quantification of MorphoIogicaI Changes In confluent cultures of astrocytes it is difficult to determine the number of cells that undergo morphological changes. As an alternative approach to directly estimating the extent of morphological changes of cells in these cultures, we monitored the shape of the intermediate filament cytoskeleton as defined by the distribution of GFAP. For these studies, cultures were fixed with methanol 1 hr; then randomly selected areas (approximately 5 mm diameter circles made with a PAP pen; Research Products International Corp., Mount Prospect, IL) approximately halfway between the center and edge of the culture dish were stained for GFAP by using the peroxidase-antiperoxidase technique (Dako PAP kit staining for GFAP; Dakopatts, Carpinteria, CA) and counterstained for nuclei with Mayer's Hematoxylin solution. From color photographs of the stained areas, the total number of nuclei in the photographed field (approximately 200 cellsifield) was counted, as was the number of cells that stained for GFAP and appeared to have processes longer than the diameter of the cell body. A percentage was then calculated by dividing the number of GFAP-positive cells with processes by the total number of cells as determined by the number of nuclei. It is important to note that at least 95% of the cells in the cultures were astrocytes, and stained for GFAP.
RESULTS Initial studies were conducted to determine the time course of the PMA-induced changes in astrocyte morphology. Cultures were exposed to 300 nM PMA for various time periods and then fixed, and the number of process-bearing cells was determined. Maximum changes in morphology were observed within 1-2 hr (Fig. 1). The number of process-bearing cells in vehicletreated (0.25 pUml DMSO) cultures ranged from 5-20% in these experiments. With PMA treatment, an additional 30-50% of the cells underwent morphological change,
*O
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1
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30
60
90
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Time (mid Fig. 1 . Time course of PMA-induced changes in astrocyte morphology. Cultures were treated with 300 nM PMA for various time periods and then fixed, and the number of process-bearing cells was determined (see Methods). Points represent means +- SE ( n = 4 ) of the number of process-bearing cells expressed as percent of the total number of cells. The two curves represent two separate experiments.
although in some batches of cells the response was greater. The half-maximal effect was calculated to occur in approximately 30 and 45 min in two different experiments (Fig. 1 ), respectively, suggesting some variation in the time course of the response from one batch of cells to another. Next, the dose-response relationship for PMA-induced changes in astrocyte morphology was determined. Various doses of PMA were added to cultures and following a 1 hr exposure period cultures were fixed and the number of process-bearing cells was determined. These studies indicated that 100 nM PMA elicits maximal changes in astrocyte morphology and half-maximal effects were observed at approximately 60 nM (Fig. 2). Studies were then conducted to determine the doseresponse relationship for the PMA-induced activation of PK-C. Cells were exposed to various doses of PMA for 10 min and PK-C activity was measured in the cytosol and membrane fractions. These studies indicate that almost all of the PK-C activity is lost from the cytosol with a PMA concentration of 50-100 nM; the half-maximal effective concentration was calculated to be 27 nM PMA (PK-C activity in the soluble fraction expressed as percent of vehicle control: 10 nM PMA, 100 ? 19%,n = 6 ; 50 nM PMA, 13 2 27%. n = 6 ; 100 nM PMA, 5 k 13%, n = 5 ; 300 nM PMA, 16 t 9%, n = 6 ) . In some batches of cells all of the PK-C activity lost from the cytosol was recovered in the particulate fraction. In other batches of
74
Harrison and Mobley 50
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2
3
Log Dose PMA (nM) Fig. 2. Dose-response relationship for PMA-induced changes in astrocyte morphology. Cultures were treated with various doses of PMA for 1 hr and then fixed, and the number of process-bearing cells was determined (see Methods). Points represent means _C SE (n = 4) of the number of process-bearing cells expressed as percent of the total number ofcells. The two curves represent two separate experiments. The single points at zero concentration represent the percent of process-bearing cells in cultures exposed to the vehicle DMSO (0.25 pl/ml) for the two experiments. cells there was an overall loss of PK-C activity from the system with this 10 min exposure period. The average loss of total PK-C activity at higher concentrations of PMA was approximately 50%. In conjunction with these studies on the effect of PMA on the distribution of PK-C, the effect of PMA was also determined on protein phosphorylation. Cells were prelabeled with 32P and then exposed to various concentrations of PMA (10,50, 100, and 300 nM) or the vehicle for I5 min. The relative incorporation of 32P into several proteins was determined. These included GFAP, vimentin, an acidic 80,000 molecular weight protein (PI 4.5), and a 30,000 molecular weight protein (PI 5.5). Maximal changes in phosphorylation were observed with 100 nM PMA (% of control; 80,000 mol.wt., 231 +- 7%; 30,000 mol.wt., 433 +- 102%; GFAP, 194 k 26; vimentin, 132 8; n = 5). Half-maximal effective concentrations of PMA were calculated from dose-response curves of data combined from three separate experiments (n = 5-6), and were as follows: GFAP, 25 nM; vimentin, 34 nM; 80,000 mol.wt., 13 nM; 30,000 mol.wt., 13 nM. Although PMA induces rapid changes in astrocyte morphology these changes are not permanent. Within 24 hr after the initial exposure the cells had almost com-
*
pletely reverted to their original shape as judged by microscopic observation using phase-contrast, and an additional treatment with 300 nM PMA at this point did not elicit further changes in morphology. To determine if the morphological response to PMA returned and, if so, when, cultures were exposed to 300 nM PMA and then 24 hr later the medium was changed to remove any remaining PMA in solution, with additional medium changes occurring every other day thereafter. The cultures were then reexposed to 300 nM PMA 1, 3, 5, or 8 days after the initial treatment (0, 2 , 4 , or 7 days after the PMA-containing medium was removed; there was no medium change on the cells re-exposed 1 day after the initial PMA treatment). These studies indicate that the return of the morphological response to PMA was gradual such that 5-8 days were required after the initial treatment (4-7 days after the 24 hr exposure period) before reexposure to PMA once again elicited a prominent change in morphology (Fig. 3). These effects of PMA on the morphology of previously untreated astrocyte cultures, cultures pretreated with PMA for 24 hr, and 8 days following the initial addition of PMA are presented in Figure 4. Studies were then conducted to determine if the loss of responsiveness to PMA and the eventual recovery of this effect correlated with changes in PK-C activity. Cultures were treated with 300 nM PMA and after 24 hr the medium was changed as described above. After exposure to PMA for 24 hr very little, if any, PK-C activity was observed in the cultures (Fig. 5). Following the 24 hr exposure period, PK-C activity in these cultures slowly began to return over a period of days; however, the rate of recovery appeared to be variable. In one set of experiments (Fig. 5 ) the PK-C activity was 20% and 77% of control at 3 and 5 days, respectively, after the initial PMA exposure. In a separate set of experiments, the amount of PK-C activity present in cells 8 days after PMA treatment was determined to be 67.4 & 8.8% (n = 7) of control. In a third study protein kinase C activity was 47% of control 5 days after the initial exposure (Table I). The recovery of PK-C activity in these cells appears to require protein synthesis since it could be blocked by cycloheximide. Cultures treated with PMA and then 24 hr after the initial exposure treated with 1 yM cycloheximide for 4 days demonstrated only 20% of the PK-C activity observed in control (DMSO-treated) cultures, whereas PK-C activity in cultures treated with PMA and then allowed to recover without cycloheximide was 47% of control (Table I). Next, studies were conducted to compare the effect of PMA on protein phosphorylation in astrocytes following a 24 hr exposure to PMA and in cells 8 days following the initial exposure to PMA. Cultured astrocytes previously exposed to either vehicle (0.5 pl/ml DMSO) or
75
Phorbol Esters and Astrocyte Morphology
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Fig. 3. Quantification of the loss and recovery of PMA-induced alterations in astrocyte shape. Astrocytes were exposed to 300 nM PMA for 24 hr. After this initial exposure period, cultures were treated with either the vehicle (0.25 pliml DMSO; dashed lineio) or 300 nM PMA (solid line/*) for 1 hr 1, 3, 5, or 8 days after the initial treatment. Additional untreated cultures were fixed and stained 1 hr after exposure to
DMSO (0)or PMA ( 0 ) .The cultures were fixed and the number of process-bearing cells was determined (see Methods). The two graphs (A and B) represent separate experiments performed in two different batches of cultures, and points represent means -+ SE (n = 4) of the percentage of process-bearing astrocytes.
PMA were labeled with 32P, re-exposed to either the vehicle or 300 nM PMA for 15 min, and then prepared for and subjected to 2-dimension gel electrophoresis. In vehicle-treated cells, a 15 min exposure to 300 nM PMA 1 day after initial DMSO treatment increased the 32P incorporation into several proteins including an acidic 80,000 molecular weight protein (PI 4 . 3 , two 30,000 molecular weight proteins (PI 5.7 and 5 . 5 ) , as well as GFAP and vimentin. This effect is shown for the 80,000 and 30,000 molecular weight (PI 5.5) proteins (Fig. 6). Exposure of vehicle-treated cells to PMA produced a 106.0 2 32.8% (n =6) increase in 32P incorporation into the 80,000 molecular weight protein and a 162.5 2 37.1% ( n = 6 ) increase in 32P incorporation into the 30,000 molecule weight protein. These changes were similar to those observed in previously untreated cells (data not shown). In contrast, no significant increase in phosphorylation was observed for either protein in PMApretreated cells exposed to a 15 min PMA treatment (109.5 ? 12.5% and 73.8 ? 8.5% for the 80,000 and 30,000 molecular weight proteins, respectively, compared to PMA-pretreated cells re-exposed to vehicle; n = 6). Similarly, no increases in the phosphorylation of vimentin or GFAP were observed in PMA-pretreated cells exposed to PMA (data not shown). The lack of PMA-induced phosphorylation in PMA-pretreated cells corresponds to the data demonstrating that little PK-C activity is present in the cultures at this time. Also, phos-
phorylation of GFAP, vimentin, and the 30,000 molecular weight proteins in vehicle-pretreated cultures exposed to DMSO for 15 min was similar to that of PMA pretreated cells exposed to DMSO. However, there was a decrease (20-40%) in the amount of phosphorylation of the 80,000 molecular weight protein in cells pretreated with PMA and exposed to DMSO, as compared to vehicle-pretreated cells exposed to DMSO (data not shown). Eight days following the initial treatment with PMA, a second PMA treatment once again produced increases in the 32P incorporation into the 80,000 and 30,000 molecular weight proteins (Fig. 7). The phosphorylation of the 80,000 molecular weight protein was increased 90.8 2 16.5% ( n = 6 ) by PMA treatment (compared to vehicle treatment) 8 days after pretreatment with DMSO. A similar increase (100.8 13.9%, n = 5 ) was observed with PMA treatment (compared to vehicle treatment) 8 days after pretreatment with PMA. A similar effect was observed for the 30,000 molecular weight protein such that an 87.5 29.5% ( n = 6 ) increase in phosphorylation was observed in cultures with PMA treatment 8 days after being pretreated with DMSO as was a 124.7 2 46.9% (n = 6) increase in cells pretreated with PMA. The phosphorylation pattern of the proteins in DMSO-pretreated cells exposed to vehicle (on day 8) was the same as that shown for PMA-pretreated cells exposed to vehicle (data not shown).
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Harrison and Mobley
DMSO
PMA
control
1 day after PMA treatment
8 days after PMA treatment
Fig. 4. The loss and recovery of PMA-induced changes in astrocyte morphology. Astrocyte cultures were treated with vehicle (0.25 pliml DMSO) or 300 nM PMA for 1 hr (top row). Additional cultures were treated for 24 hr with 300 nM PMA and then exposed to DMSO or PMA for 1 hr (middle
row), or treated with PMA for 24 hr and then reexposed 8 days after the initial treatment to either DMSO or PMA for 1 hr (bottom row). (Photographs are of methanol-fixed cells prior to staining for GFAP.) Magnification = 100 X .
DISCUSSION
unclear if the effect of the phorbol ester on cell shape correlated with the activation of PK-C and subsequent protein phosphorylation. In the present studies two approaches were used to examine the role of the kinase system in mediating the effects of PMA on astrocyte morphology. The first was to compare the dose-response relationship between the effects of PMA on astrocyte morphology, the distribution of PK-C to the membrane, and protein phosphorylation.
In previous studies we have demonstrated that 300 nM PMA can induce dramatic changes in the morphology of cultured astrocytes, and can also shift PK-C activity from the soluble to particulate fraction (Mobley et al., 1986). Additionally, we have demonstrated that substantial changes in protein phosphorylation occur within 15 min and precede the changes in morphology (Harrison and Mobley, 1989: present results). However, it was
Phorbol Esters and Astrocyte Morphology
77
quired to elicit a half-maximum change in astrocyte morphology was calculated to be approximately 60 nM. This appears to be slightly higher than that required for the activation of PK-C. Whether this is the consequence of the inefficient coupling of phosphorylation events mediated by PK-C and the biochemical events which actually underlie the changes in cell shape, the morphological effect being mediated by a small pool of PK-C which requires higher PMA concentrations for its activation, or other factors remains to be determined. A second approach to evaluate the role of PK-C in regulating astrocyte morphology was with the use of astrocytes depleted of this kinase by preexposure to PMA. Treatment with phorbol esters has been shown to deplete PK-C in several cells types (Hepler et al., 1988; Darbon 0 I 2 3 4 5 et al., 1987; Chida et al., 1986; Kraft et al., 1982), and Days in preliminary studies we have observed a similar pheFig. 5 . PMA-induced loss of PK-C activity and subsequent nomenon in astrocytes (Harrison and Mobley, 1987). recovery of kinase activity. Astrocyte cultures were exposed to Although PMA produces rapid changes in the morpholeither vehicle (0.25 pliml DMSO) or 300 nM PMA for 24 hr. ogy of cultured astrocytes, the cells have returned back Some cultures were assayed for PK-C activity at this time. Other cultures were placed in fresh medium and then assayed almost to their original shape within 24 hr and these cells for PK-C activity 3 and 5 days after the initial PMA treatment. lack measurable PK-C activity. A second exposure to The results (means ? SE, n = 6) represent the percent of the PMA at this time fails to produce any further changes in total amount of PK-C activity as compared to untreated control cell shape. The loss of PK-C activity in some types of cultures. Treatment with the DMSO vehicle had no effect on cells has previously been demonstrated to be due to an protein kinase C activity. increased rate of degradation of the enzyme by a Ca’ activated neutral protease (Kajikawa et a]., 1983; Young TABLE 1. Cycloheximide Inhibition of the Recovery of Protein et al., 1987). Whether a similar mechanism accounts for Kinase C in Astrocytes Pretreated With Either DMSO or PMA the loss of PK-C in astrocytes remains to be determined. for 24 Hrt Accompanying this loss of PK-C was also a loss in PK-C activity the ability of PMA to induce changes in protein phosTreatment (% control) phorylation. In untreated cells or DMSO-treated cells, 100.00 * 10.29 PMA can elicit pronounced increases in the j2P content DMSO DMSO + cycloheximide 61.07 * 5.48 of several proteins including GFAP, vimentin, an acidic 46.89 -t 2.49 PMA 80,000 molecular weight protein, and two 30,000 nioPMA + cycloheximide 22.35 2 1.68” lecular weight proteins (Harrison and Mobley, 1989). In TAstrocytes were treated with vehicle (0.025% DMSO) or 300 nM cells preexposed to PMA for 24 hr the phosphorylation PMA for 24 hr. At this time the medium was changcd and cyclohexresponse is lost. The observation that PMA is no longer imide ( 1 p M final concentration) was added daily to somc cultures for the next 4 days. The cultures were then assayed for total PK-C activ- capable of increasing the ”P incorporation into proteins when cells are depleted of PK-C supports the idea that ity. The values represent the mean +- SE (n = 10). *P
Phorbol Ester-Induced Change in Astrocyte Morphology: Correlation With Protein Kinase C Activation and Protein Phosphorylation B.C. Harrison and P.L. Mobley Department of Pharmacology, University of Texas Health Science Center, San Antonio
Treatment with 300 nM phorbol 12-myristate 13-acetate (PMA) transforms polygonal-shaped cultured astrocytes into process-bearing cells and produces a shift in protein kinase C (PK-C) from the cytosol to the membrane. Exposure to PMA also produces increases in the phosphorylation of several proteins including vimentin, glial fibrillary acidic protein (GFAP), an acidic 80,000 molecular weight protein, and two 30,000 molecular weight proteins (PI5.5 and 5.7). The effects of PMA on the translocation of PK-C and on protein phosphorylation precede the PMAinduced changes in astrocyte morphology, and a close correlation exists between the concentration of PMA necessary to elicit half-maximal and maximal effects on the shift of PK-C to the membrane and on protein phosphorylation. In addition, the PMA-induced alterations in cell morphology are not permanent, and within 24 hr after PMA treatment the cells have reverted almost to their original morphology. A second exposure to PMA at this time fails to elicit further change in cell shape and is also incapable of producing increases in the phosphorylation of proteins. It was determined that there is little, if any, PK-C present in these PMA-pretreated cells. The morphological responsiveness to PMA gradually returns in 5 to 8 days after the initial treatment with PMA, and this is accompanied by the recovery of PK-C activity and the phosphorylation response. Therefore, these studies suggest that the effect of PMA on astrocyte morphology is mediated by the activation of PK-C and subsequent protein phosphorylation. Key words: glia, GFAP, vimentin
be conducted across the cell membrane to regulate cell function. One such system involves the generation of inositol trisphosphate and diacylglycerol from the hydrolysis of phosphatidylinositol (Friedel et al., 1969; Berridge, 1984; Nishizuka, 1984). lnositol trisphosphate promotes the release of calcium from intracellular stores (Streb et al., 1983; Berridge and Irvine, 1984) while diacylglycerol is capable of activating the calcium and phospholipid-dependent protein kinase, protein kinase C (PK-C) (Takai et al., 1979; Kishimoto et al., 1980). PK-C appears to have a ubiquitous distribution in animal tissue and has been reported to be present in cultures of rat astrocytes (Mobley et al., 1986; Neary et al., 1986). It has also been demonstrated that the PK-C activator, phorbol 12-myristate 13-acetate (PMA), produces changes in astrocyte morphology such that flat, polygonal-shaped cells are transformed into stellate, process-bearing cells (Mobley et al., 1986). PMA has also been reported to change the morphology of other cell types including fibroblasts (Diamond et al., 1974), the epithelial cell line MDCK (Ojakian, 1981), and macrophages (Phaire-Washington et al., 1980). Besides eliciting alterations in cell shape, treatment of astrocyte cultures with PMA was found to produce a rapid translocation of PK-C activity from the cytosol to the membrane of these cells (Mobley et al., 1986; Neary et al., 1988). Previous studies have also demonstrated that treatment of astrocytes with 300 nM PMA produces increases in the 32P incorporation into several phosphoproteins including an 80,000 (PI 4.5) and two 30,000 (PI 5.5 and 5.7) molecular weight proteins and the cytoskeletal proteins glial fibrillary acidic protein (GFAP) and vimentin (Mobley and Harrison, 1987; Harrison and Mobley, 1989). While treatment of astrocytes with PMA induces
INTRODUCTION Extracellular signals such as neurotransmitters and hormones can induce the generation of signal messengers that activate protein kinases. In turn, protein kinases are capable of regulating the phosphorylation of proteins, thereby establishing a means by which information can 0 1990 Wiley-Liss, Inc.
Received July 12, 1989; revised September 14, 1989; accepted September 15, 1989. Address reprint requests to Philip L. Mobley, Ph.D., Department of Pharmacology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284.
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Harrison and Mobley
changes in astrocyte morphology and protein phosphorylation, it is not certain if these two effects are related to each other or to what extent these effects are mediated by PK-C. In the present studies we have attempted to correlate the effects of PMA on astrocyte morphology with changes in PK-C activation and subsequent protein phosphorylation. Also, the effects of PMA on morphology and protein phosphorylation have been studied in PKC-depleted astrocytes. The results indicate that the effects of PMA on astrocyte morphology correlated with a shift of PK-C to the membrane fraction and an increase in protein phosphorylation of several proteins. Additionally, in PK-C-depleted cells PMA lost its ability to induce changes in cell morphology or protein phosphorylation. A substantial amount of PK-C activity returned within 5 to 8 days after the initial exposure to PMA, and this correlates with a return of the morphological and phosphorylation response to this agent.
EGTA, 2 mM EDTA, 1 mM dithiothreitol, 0.1 mgiml leupeptin, 0. I mM phenylmethylsulfonyl fluoride, pH 7.5) and placed on ice. Cell suspensions were then centrifuged for 1 min at 120,000g in a Beckman Airfuge. The supernatant (soluble extract) was saved and the resulting pellet was resuspended in Tris buffer and centrifuged. The supernatant was discarded and the pellet was resuspended in Tris buffer 0.1% Triton X-100 and placed on ice for 30 min. The suspension was centrifuged and the resulting supernatant (membrane extract) saved. The level of PK-C activity was determined in both the soluble and membrane fractions as described previously (Kikkawa et al., 1982) by using histone (Type III-S; Sigma Chemical Co., St. Louis, MO) as a substrate. For experiments where the PMA-induced loss and subsequent recovery of total PK-C activity was determined, cells were homogenized directly in buffer containing 0.1% Triton X- 100 and the total homogenate was assayed for PK-C. The total assay volume was 50 pl and contained (final concentration) 20 mM Tris-HC1 (pH MATERIALS AND METHODS 7 . 3 , 2 mM EGTA, 0.4 mM EDTA, 0.2 mM dithiothreCell Culture itol, 20 pg/ml leupeptin, 0.02 mM phenylmethylsulfonyl P , pg histone, and 10 (1.1 Astrocyte cultures were prepared from the neocor- fluoride, 100 pM [ Y - ~ ~ P I - A T40 tex of 1 day-old Sprague-Dawley rats. The tissue was tissue preparation. The reaction was conducted at 30°C sliced at 400 pm intervals with a McIlwain Tissue Chop- for 3 min in the presence or absence of 1.5 mM CaCI,, per and placed in sterile Krebs Ringer bicarbonate buffer 3 mM Mg-acetate, 20 p g phosphatidylserine, and 0.3 pg (pH 7.4) with trypsin (2500 unitsiml) for 5 min at 37°C. diolein (98% 1,2 isomer; Sigma Chemical Co., St. Medium containing fetal bovine serum was then added to Louis, MO). Aliquots of the reaction mixture were inhibit the trypsin. The cells were pelleted and then trit- placed on filter paper squares ( 1 .S cm; Whatman 3MM) urated in calcium and magnesium-free Krebs Ringer bi- and immersed in 10% trichloroacetic acid (TCA) to tercarbonate buffer containing 0.0 1 mgiml DNase (deoxy- minate the reaction. After washing the filter paper ribonuclease 1 ; Boehringer Mannheim, Indianapolis, squares in boiling 5% TCA (twice), ethanol, and acetone IN). The dispersed cells were transferred to Dulbecco’s (twice), radioactivity was determined by Cerenkov modified Eagle’s medium with high glucose (DMEM; counting in water. pH 7.4) containing 10% fetal bovine serum and then filtered through a 70 pm nylon screen and plated (300,000 cells/cm2) in tissue culture flasks (25 cm2). The Protein Phosphorylation Confluent cultures were washed with sterile 0.9% astrocytes were grown to confluence in DMEM plus serum in an atmosphere of 95% air/S% CO, at 37°C. NaCl followed by phosphate-free DMEM, and then 1 ml Contaminating cells were removed by orbital shaking of phosphate-free DMEM containing 5% dialyzed calf (McCarthy and de Vellis, 1980) and the astrocytes were serum was added to each dish. An aliquot of ’2P-orthosubcultured into 35 mm dishes (30,000 cells/cm2) and phosphate (0.1-0.2 mCi; approximately 285 Ciimg) was maintained as described above. Based on morphology added to each dish and after a 2 hr labeling period, the and immunocytochemical studies (peroxidase-antiperox- cultures were exposed to either 300 nM PMA or the idase staining for GFAP) these cultures were relatively dimethyl sulfoxide vehicle (DMSO, 0.05%) for 1.5 min. free of contaminating cells (greater than 9.5% astrocytes). After treatment, the culture medium was removed and Cultures were then used for experiments within 14 days the cells were washed once with ice-cold PBS and then dissolved in 1% sodium dodecyl sulfate (SDS). An aliafter reaching confluence. quot of this solution was then added to 9 volumes of Protein Kinase C Assay ice-cold acetone. The precipitate was collected by cenThe culture medium was removed and the cells trifugation and dissolved in urea-containing lysis buffer were washed twice with ice-cold phosphate-buffered sa- (O’Farrell, 1975). Equal volumes (30 pl; approximately line (PBS). The cells were scraped from the dishes and 60 pg protein) of the samples were then subjected to homogenized in Tris buffer (20 mM Tris-HCI, 10 mM 2-dimension gel electrophoresis (O’Farrell, 1975). The
+
Phorbol Esters and Gstrocyte Morphology
protein content of the culture dishes and 32P incorporation are quite uniform such that the samples contained approximately equal amounts of protein and acidiorganic solvent insoluble radioactivity. After electrophoresis, the gels were fixed in a solution of 10% acetic acid and 10% isopropanol and dried, and autoradiograms were prepared by using x-ray film (Kodak XAR-5) with or without Dupont Lightning Plus intensifying screens as necessary. Computer images of the protein spots on the autoradiograms were made by using a Grinnell imageprocessing system coupled to a VAX-111750 computer. The degree of exposure of each spot of interest was determined based on the gray level of each pixel that comprised the spot image.
Quantification of MorphoIogicaI Changes In confluent cultures of astrocytes it is difficult to determine the number of cells that undergo morphological changes. As an alternative approach to directly estimating the extent of morphological changes of cells in these cultures, we monitored the shape of the intermediate filament cytoskeleton as defined by the distribution of GFAP. For these studies, cultures were fixed with methanol 1 hr; then randomly selected areas (approximately 5 mm diameter circles made with a PAP pen; Research Products International Corp., Mount Prospect, IL) approximately halfway between the center and edge of the culture dish were stained for GFAP by using the peroxidase-antiperoxidase technique (Dako PAP kit staining for GFAP; Dakopatts, Carpinteria, CA) and counterstained for nuclei with Mayer's Hematoxylin solution. From color photographs of the stained areas, the total number of nuclei in the photographed field (approximately 200 cellsifield) was counted, as was the number of cells that stained for GFAP and appeared to have processes longer than the diameter of the cell body. A percentage was then calculated by dividing the number of GFAP-positive cells with processes by the total number of cells as determined by the number of nuclei. It is important to note that at least 95% of the cells in the cultures were astrocytes, and stained for GFAP.
RESULTS Initial studies were conducted to determine the time course of the PMA-induced changes in astrocyte morphology. Cultures were exposed to 300 nM PMA for various time periods and then fixed, and the number of process-bearing cells was determined. Maximum changes in morphology were observed within 1-2 hr (Fig. 1). The number of process-bearing cells in vehicletreated (0.25 pUml DMSO) cultures ranged from 5-20% in these experiments. With PMA treatment, an additional 30-50% of the cells underwent morphological change,
*O
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1
0
30
60
90
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Time (mid Fig. 1 . Time course of PMA-induced changes in astrocyte morphology. Cultures were treated with 300 nM PMA for various time periods and then fixed, and the number of process-bearing cells was determined (see Methods). Points represent means +- SE ( n = 4 ) of the number of process-bearing cells expressed as percent of the total number of cells. The two curves represent two separate experiments.
although in some batches of cells the response was greater. The half-maximal effect was calculated to occur in approximately 30 and 45 min in two different experiments (Fig. 1 ), respectively, suggesting some variation in the time course of the response from one batch of cells to another. Next, the dose-response relationship for PMA-induced changes in astrocyte morphology was determined. Various doses of PMA were added to cultures and following a 1 hr exposure period cultures were fixed and the number of process-bearing cells was determined. These studies indicated that 100 nM PMA elicits maximal changes in astrocyte morphology and half-maximal effects were observed at approximately 60 nM (Fig. 2). Studies were then conducted to determine the doseresponse relationship for the PMA-induced activation of PK-C. Cells were exposed to various doses of PMA for 10 min and PK-C activity was measured in the cytosol and membrane fractions. These studies indicate that almost all of the PK-C activity is lost from the cytosol with a PMA concentration of 50-100 nM; the half-maximal effective concentration was calculated to be 27 nM PMA (PK-C activity in the soluble fraction expressed as percent of vehicle control: 10 nM PMA, 100 ? 19%,n = 6 ; 50 nM PMA, 13 2 27%. n = 6 ; 100 nM PMA, 5 k 13%, n = 5 ; 300 nM PMA, 16 t 9%, n = 6 ) . In some batches of cells all of the PK-C activity lost from the cytosol was recovered in the particulate fraction. In other batches of
74
Harrison and Mobley 50
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Log Dose PMA (nM) Fig. 2. Dose-response relationship for PMA-induced changes in astrocyte morphology. Cultures were treated with various doses of PMA for 1 hr and then fixed, and the number of process-bearing cells was determined (see Methods). Points represent means _C SE (n = 4) of the number of process-bearing cells expressed as percent of the total number ofcells. The two curves represent two separate experiments. The single points at zero concentration represent the percent of process-bearing cells in cultures exposed to the vehicle DMSO (0.25 pl/ml) for the two experiments. cells there was an overall loss of PK-C activity from the system with this 10 min exposure period. The average loss of total PK-C activity at higher concentrations of PMA was approximately 50%. In conjunction with these studies on the effect of PMA on the distribution of PK-C, the effect of PMA was also determined on protein phosphorylation. Cells were prelabeled with 32P and then exposed to various concentrations of PMA (10,50, 100, and 300 nM) or the vehicle for I5 min. The relative incorporation of 32P into several proteins was determined. These included GFAP, vimentin, an acidic 80,000 molecular weight protein (PI 4.5), and a 30,000 molecular weight protein (PI 5.5). Maximal changes in phosphorylation were observed with 100 nM PMA (% of control; 80,000 mol.wt., 231 +- 7%; 30,000 mol.wt., 433 +- 102%; GFAP, 194 k 26; vimentin, 132 8; n = 5). Half-maximal effective concentrations of PMA were calculated from dose-response curves of data combined from three separate experiments (n = 5-6), and were as follows: GFAP, 25 nM; vimentin, 34 nM; 80,000 mol.wt., 13 nM; 30,000 mol.wt., 13 nM. Although PMA induces rapid changes in astrocyte morphology these changes are not permanent. Within 24 hr after the initial exposure the cells had almost com-
*
pletely reverted to their original shape as judged by microscopic observation using phase-contrast, and an additional treatment with 300 nM PMA at this point did not elicit further changes in morphology. To determine if the morphological response to PMA returned and, if so, when, cultures were exposed to 300 nM PMA and then 24 hr later the medium was changed to remove any remaining PMA in solution, with additional medium changes occurring every other day thereafter. The cultures were then reexposed to 300 nM PMA 1, 3, 5, or 8 days after the initial treatment (0, 2 , 4 , or 7 days after the PMA-containing medium was removed; there was no medium change on the cells re-exposed 1 day after the initial PMA treatment). These studies indicate that the return of the morphological response to PMA was gradual such that 5-8 days were required after the initial treatment (4-7 days after the 24 hr exposure period) before reexposure to PMA once again elicited a prominent change in morphology (Fig. 3). These effects of PMA on the morphology of previously untreated astrocyte cultures, cultures pretreated with PMA for 24 hr, and 8 days following the initial addition of PMA are presented in Figure 4. Studies were then conducted to determine if the loss of responsiveness to PMA and the eventual recovery of this effect correlated with changes in PK-C activity. Cultures were treated with 300 nM PMA and after 24 hr the medium was changed as described above. After exposure to PMA for 24 hr very little, if any, PK-C activity was observed in the cultures (Fig. 5). Following the 24 hr exposure period, PK-C activity in these cultures slowly began to return over a period of days; however, the rate of recovery appeared to be variable. In one set of experiments (Fig. 5 ) the PK-C activity was 20% and 77% of control at 3 and 5 days, respectively, after the initial PMA exposure. In a separate set of experiments, the amount of PK-C activity present in cells 8 days after PMA treatment was determined to be 67.4 & 8.8% (n = 7) of control. In a third study protein kinase C activity was 47% of control 5 days after the initial exposure (Table I). The recovery of PK-C activity in these cells appears to require protein synthesis since it could be blocked by cycloheximide. Cultures treated with PMA and then 24 hr after the initial exposure treated with 1 yM cycloheximide for 4 days demonstrated only 20% of the PK-C activity observed in control (DMSO-treated) cultures, whereas PK-C activity in cultures treated with PMA and then allowed to recover without cycloheximide was 47% of control (Table I). Next, studies were conducted to compare the effect of PMA on protein phosphorylation in astrocytes following a 24 hr exposure to PMA and in cells 8 days following the initial exposure to PMA. Cultured astrocytes previously exposed to either vehicle (0.5 pl/ml DMSO) or
75
Phorbol Esters and Astrocyte Morphology
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Fig. 3. Quantification of the loss and recovery of PMA-induced alterations in astrocyte shape. Astrocytes were exposed to 300 nM PMA for 24 hr. After this initial exposure period, cultures were treated with either the vehicle (0.25 pliml DMSO; dashed lineio) or 300 nM PMA (solid line/*) for 1 hr 1, 3, 5, or 8 days after the initial treatment. Additional untreated cultures were fixed and stained 1 hr after exposure to
DMSO (0)or PMA ( 0 ) .The cultures were fixed and the number of process-bearing cells was determined (see Methods). The two graphs (A and B) represent separate experiments performed in two different batches of cultures, and points represent means -+ SE (n = 4) of the percentage of process-bearing astrocytes.
PMA were labeled with 32P, re-exposed to either the vehicle or 300 nM PMA for 15 min, and then prepared for and subjected to 2-dimension gel electrophoresis. In vehicle-treated cells, a 15 min exposure to 300 nM PMA 1 day after initial DMSO treatment increased the 32P incorporation into several proteins including an acidic 80,000 molecular weight protein (PI 4 . 3 , two 30,000 molecular weight proteins (PI 5.7 and 5 . 5 ) , as well as GFAP and vimentin. This effect is shown for the 80,000 and 30,000 molecular weight (PI 5.5) proteins (Fig. 6). Exposure of vehicle-treated cells to PMA produced a 106.0 2 32.8% (n =6) increase in 32P incorporation into the 80,000 molecular weight protein and a 162.5 2 37.1% ( n = 6 ) increase in 32P incorporation into the 30,000 molecule weight protein. These changes were similar to those observed in previously untreated cells (data not shown). In contrast, no significant increase in phosphorylation was observed for either protein in PMApretreated cells exposed to a 15 min PMA treatment (109.5 ? 12.5% and 73.8 ? 8.5% for the 80,000 and 30,000 molecular weight proteins, respectively, compared to PMA-pretreated cells re-exposed to vehicle; n = 6). Similarly, no increases in the phosphorylation of vimentin or GFAP were observed in PMA-pretreated cells exposed to PMA (data not shown). The lack of PMA-induced phosphorylation in PMA-pretreated cells corresponds to the data demonstrating that little PK-C activity is present in the cultures at this time. Also, phos-
phorylation of GFAP, vimentin, and the 30,000 molecular weight proteins in vehicle-pretreated cultures exposed to DMSO for 15 min was similar to that of PMA pretreated cells exposed to DMSO. However, there was a decrease (20-40%) in the amount of phosphorylation of the 80,000 molecular weight protein in cells pretreated with PMA and exposed to DMSO, as compared to vehicle-pretreated cells exposed to DMSO (data not shown). Eight days following the initial treatment with PMA, a second PMA treatment once again produced increases in the 32P incorporation into the 80,000 and 30,000 molecular weight proteins (Fig. 7). The phosphorylation of the 80,000 molecular weight protein was increased 90.8 2 16.5% ( n = 6 ) by PMA treatment (compared to vehicle treatment) 8 days after pretreatment with DMSO. A similar increase (100.8 13.9%, n = 5 ) was observed with PMA treatment (compared to vehicle treatment) 8 days after pretreatment with PMA. A similar effect was observed for the 30,000 molecular weight protein such that an 87.5 29.5% ( n = 6 ) increase in phosphorylation was observed in cultures with PMA treatment 8 days after being pretreated with DMSO as was a 124.7 2 46.9% (n = 6) increase in cells pretreated with PMA. The phosphorylation pattern of the proteins in DMSO-pretreated cells exposed to vehicle (on day 8) was the same as that shown for PMA-pretreated cells exposed to vehicle (data not shown).
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Harrison and Mobley
DMSO
PMA
control
1 day after PMA treatment
8 days after PMA treatment
Fig. 4. The loss and recovery of PMA-induced changes in astrocyte morphology. Astrocyte cultures were treated with vehicle (0.25 pliml DMSO) or 300 nM PMA for 1 hr (top row). Additional cultures were treated for 24 hr with 300 nM PMA and then exposed to DMSO or PMA for 1 hr (middle
row), or treated with PMA for 24 hr and then reexposed 8 days after the initial treatment to either DMSO or PMA for 1 hr (bottom row). (Photographs are of methanol-fixed cells prior to staining for GFAP.) Magnification = 100 X .
DISCUSSION
unclear if the effect of the phorbol ester on cell shape correlated with the activation of PK-C and subsequent protein phosphorylation. In the present studies two approaches were used to examine the role of the kinase system in mediating the effects of PMA on astrocyte morphology. The first was to compare the dose-response relationship between the effects of PMA on astrocyte morphology, the distribution of PK-C to the membrane, and protein phosphorylation.
In previous studies we have demonstrated that 300 nM PMA can induce dramatic changes in the morphology of cultured astrocytes, and can also shift PK-C activity from the soluble to particulate fraction (Mobley et al., 1986). Additionally, we have demonstrated that substantial changes in protein phosphorylation occur within 15 min and precede the changes in morphology (Harrison and Mobley, 1989: present results). However, it was
Phorbol Esters and Astrocyte Morphology
77
quired to elicit a half-maximum change in astrocyte morphology was calculated to be approximately 60 nM. This appears to be slightly higher than that required for the activation of PK-C. Whether this is the consequence of the inefficient coupling of phosphorylation events mediated by PK-C and the biochemical events which actually underlie the changes in cell shape, the morphological effect being mediated by a small pool of PK-C which requires higher PMA concentrations for its activation, or other factors remains to be determined. A second approach to evaluate the role of PK-C in regulating astrocyte morphology was with the use of astrocytes depleted of this kinase by preexposure to PMA. Treatment with phorbol esters has been shown to deplete PK-C in several cells types (Hepler et al., 1988; Darbon 0 I 2 3 4 5 et al., 1987; Chida et al., 1986; Kraft et al., 1982), and Days in preliminary studies we have observed a similar pheFig. 5 . PMA-induced loss of PK-C activity and subsequent nomenon in astrocytes (Harrison and Mobley, 1987). recovery of kinase activity. Astrocyte cultures were exposed to Although PMA produces rapid changes in the morpholeither vehicle (0.25 pliml DMSO) or 300 nM PMA for 24 hr. ogy of cultured astrocytes, the cells have returned back Some cultures were assayed for PK-C activity at this time. Other cultures were placed in fresh medium and then assayed almost to their original shape within 24 hr and these cells for PK-C activity 3 and 5 days after the initial PMA treatment. lack measurable PK-C activity. A second exposure to The results (means ? SE, n = 6) represent the percent of the PMA at this time fails to produce any further changes in total amount of PK-C activity as compared to untreated control cell shape. The loss of PK-C activity in some types of cultures. Treatment with the DMSO vehicle had no effect on cells has previously been demonstrated to be due to an protein kinase C activity. increased rate of degradation of the enzyme by a Ca’ activated neutral protease (Kajikawa et a]., 1983; Young TABLE 1. Cycloheximide Inhibition of the Recovery of Protein et al., 1987). Whether a similar mechanism accounts for Kinase C in Astrocytes Pretreated With Either DMSO or PMA the loss of PK-C in astrocytes remains to be determined. for 24 Hrt Accompanying this loss of PK-C was also a loss in PK-C activity the ability of PMA to induce changes in protein phosTreatment (% control) phorylation. In untreated cells or DMSO-treated cells, 100.00 * 10.29 PMA can elicit pronounced increases in the j2P content DMSO DMSO + cycloheximide 61.07 * 5.48 of several proteins including GFAP, vimentin, an acidic 46.89 -t 2.49 PMA 80,000 molecular weight protein, and two 30,000 nioPMA + cycloheximide 22.35 2 1.68” lecular weight proteins (Harrison and Mobley, 1989). In TAstrocytes were treated with vehicle (0.025% DMSO) or 300 nM cells preexposed to PMA for 24 hr the phosphorylation PMA for 24 hr. At this time the medium was changcd and cyclohexresponse is lost. The observation that PMA is no longer imide ( 1 p M final concentration) was added daily to somc cultures for the next 4 days. The cultures were then assayed for total PK-C activ- capable of increasing the ”P incorporation into proteins when cells are depleted of PK-C supports the idea that ity. The values represent the mean +- SE (n = 10). *P
